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Tumor reliance on glycolysis is a hallmark of cancer. Immunotherapy is more effective in controlling glycolysis-low tumors lacking lactate dehydrogenase (LDH) due to reduced tumor lactate efflux and enhanced glucose availability within the tumor microenvironment (TME). LDH inhibitors (LDHi) reduce glucose uptake and tumor growth in preclinical models, but their impact on tumor-infiltrating T cells is not fully elucidated. Tumor cells have higher basal LDH expression and glycolysis levels compared with infiltrating T cells, creating a therapeutic opportunity for tumor-specific targeting of glycolysis. We demonstrate that LDHi treatment (a) decreases tumor cell glucose uptake, expression of the glucose transporter GLUT1, and tumor cell proliferation while (b) increasing glucose uptake, GLUT1 expression, and proliferation of tumor-infiltrating T cells. Accordingly, increasing glucose availability in the microenvironment via LDH inhibition leads to improved tumor-killing T cell function and impaired Treg immunosuppressive activity in vitro. Moreover, combining LDH inhibition with immune checkpoint blockade therapy effectively controls murine melanoma and colon cancer progression by promoting effector T cell infiltration and activation while destabilizing Tregs. Our results establish LDH inhibition as an effective strategy for rebalancing glucose availability for T cells within the TME, which can enhance T cell function and antitumor immunity.

Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. Although immunotherapy is being explored as a potential treatment option for patients with GBM, it is unclear whether systemic immunotherapy can reach and modify the tumor microenvironment in the brain. We evaluated immune characteristics in patients receiving the anti-PD-1 immune checkpoint inhibitor nivolumab 1 week prior to surgery, compared with control patients receiving salvage resection without prior nivolumab treatment. We observed saturating levels of nivolumab bound to intratumorally and tissue-resident T cells in the brain, implicating saturating levels of nivolumab reaching brain tumors. Following nivolumab treatment, significant changes in T-cell activation and proliferation were observed in the tumor-resident T-cell population, and peripheral T cells upregulated chemokine receptors related to brain homing. A strong nivolumab-driven upregulation in compensatory checkpoint inhibition molecules, i.e., TIGIT, LAG-3, TIM-3, and CTLA-4, was observed, potentially counteracting the treatment effect. Finally, tumor-reactive tumor-infiltrating lymphocytes (TIL) were found in a subset of nivolumab-treated patients with prolonged survival, and neoantigen-reactive T cells were identified in both TILs and blood. This indicates a systemic response toward GBM in a subset of patients, which was further boosted by nivolumab, with T-cell responses toward tumor-derived neoantigens. Our study demonstrates that nivolumab does reach the GBM tumor lesion and enhances antitumor T-cell responses both intratumorally and systemically. However, various anti-inflammatory mechanisms mitigate the clinical efficacy of the anti-PD-1 treatment., (©2024 The Authors; Published by the American Association for Cancer Research.)

Non-small cell lung cancer (NSCLC) is the most pervasive lung cancer subtype. Recent studies have shown that immune checkpoint inhibitors achieved favorable clinical benefits in resectable NSCLC; however, the associated mechanism remains unclear. The role of T cells in antitumor immunity has received considerable attention, while the antitumor effects of tumor-infiltrating B cells (TIBs) in NSCLC remain poorly understood. Here, we conducted a single-cell RNA sequencing analysis of immune cells isolated from 12 patients with stage IIIA NSCLC to investigate B cell subtypes and their functions following neoadjuvant chemoimmunotherapy. We confirmed the simultaneous existence of the 4 B cell subtypes. Among them, memory B cells were found to be associated with a positive therapeutic effect to neoadjuvant chemoimmunotherapy. Furthermore, we found that G protein-coupled receptor 183 was most prevalent in memory B cells and associated with a positive therapeutic response. Multiplex immunofluorescence and flow cytometry experiments in an additional cohort of 22 treatment-naïve and 30 stage IIIA/IIIB NSCLC patients treated with neoadjuvant chemoimmunotherapy verified these findings. Overall, our analysis revealed the functions of TIBs and their potential effect on clinical treatment in NSCLC., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: We aimed to explore the effects and mechanisms of action of dehydroepiandrosterone (DHEA) on immune evasion of oral squamous cell carcinoma (OSCC) to provide evidence for enhancing the effect of immunotherapy., Materials and Methods: A xenograft mouse model and immunohistochemistry were used to reveal the patterns of tumor-infiltrating lymphocytes (TILs). The CAL27 and SCC VII cell lines were used for the in vitro study. Western blotting, qPCR, immunofluorescence, and flow cytometry were used to evaluate the expression of B7-H4. Recombinant mouse B7-H4 protein (rmB7-H4) and PG490, an inhibitor of NF-κB p65 were used for the "rescue study." Gain- and loss-of-function, luciferase reporter, and chromatin immunoprecipitation assays were performed to verify this mechanism., Results: DHEA inhibited tumor growth in an OSCC xenograft mouse model, increased CD8 + cells, and decreased FOXP3 + cells in TILs. DHEA reduced the expression of B7-H4 in CAL27 and SCC VII cells RmB7-H4 reverses the effect of DHEA on tumor growth and TIL patterns. DHEA increased the expression of miR-15b-5p and activated its transcriptional factor NF-κB p65. Further experiments demonstrated that miR-15b-5p inhibited B7-H4 expression by binding to its 3'-UTR regions, and NF-κB p65 activated miR-15b transcription. PG490 reversed the effects of DHEA on tumor growth, antitumor immunity in the OSCC xenograft model, and the expression/phosphorylation of NF-κB p65, miR-15b-5p, and B7-H4., Conclusions: This study indicates that DHEA attenuates the immune escape of OSCC cells by inhibiting B7-H4 expression, providing new insights for cancer immunotherapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yifei Wang reports financial support was provided by National Natural Science Foundation of China. Yifei Wang reports financial support was provided by Beijing Natural Science Foundation Committee. Chuanbin Guo reports financial support was provided by Ministry of Science and Technology of the People’s Republic of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

It is crucial to decipher the modulation of regulatory T cells (Tregs) in tumor microenvironment (TME) induced by chemotherapy, which may contribute to improving the efficacy of neoadjuvant chemoimmunotherapy in resectable non-small cell lung cancer (NSCLC). We retrospectively collected specimens from patients with II-III NSCLC, constituting two cohorts: a neoadjuvant chemotherapy (NAC) cohort (N = 141) with biopsy (N = 58) and postoperative specimens (N = 141), and a surgery-only cohort (N = 122) as the control group. Then, the cell density (Dens), infiltration score (InS), and Treg-cell proximity score (TrPS) were conducted using a panel of multiplex fluorescence staining (Foxp3, CD4, CD8, CK, CD31, ɑSMA). Subsequently, the association of Tregs with cancer microvessels (CMVs) and cancer-associated fibroblasts (CAFs) was analyzed. Patients with NAC treatment have a higher density of Tregs in both paired (P < 0.001) and unpaired analysis (P = 0.022). Additionally, patients with NAC treatment showed higher infiltration score (paired, P < 0.001; unpaired, P = 0.014) and more CD8 + T cells around Tregs (paired/unpaired, both P < 0.001). Subgroup analysis indicated that tumors with a diameter of ≤ 5 cm exhibited increase in both Dens (Treg) and InS (Treg) , and gemcitabine, pemetrexed and taxel enhanced Dens (Treg) and TrPS (CD8) following NAC. Multivariate analysis identified that the Dens (Tregs) , InS (Tregs) and TrPS (CD8) were significantly associated with better chemotherapy response [OR = 8.54, 95%CI (1.69, 43.14), P = 0.009; OR = 7.14, 95%CI (1.70, 30.08), P = 0.024; OR = 5.50, 95%CI (1.09, 27.75), P = 0.039, respectively] and positive recurrence-free survival [HR = 3.23, 95%CI (1.47, 7.10), P = 0.004; HR = 2.70; 95%CI (1.27, 5.72); P = 0.010; HR = 2.55, 95%CI (1.21, 5.39), P = 0.014, respectively]. Moreover, TrPS (CD8) and TrPS (CD4) were negatively correlated with the CMVs and CAFs. These discoveries have deepened our comprehension of the immune-modulating impact of chemotherapy and underscored that the modified spatial landscape of Tregs after chemotherapy should be taken into account for personalized immunotherapy, aiming to ultimately improve clinical outcomes in patients with NSCLC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

For triple-negative breast cancer (TNBC), the most aggressive subset of breast cancer, immune cell infiltrates have prognostic implications. The presence of myeloid-derived suppressor cells supports tumor progression, while tumor-infiltrating lymphocytes (TILs) correlate with improved survival and responsiveness to immunotherapy. Manipulating the abundance of these populations may enhance tumor immunity. Gemcitabine (GEM), a clinically employed chemotherapeutic, is reported to be systemically myeloablative, and thus it is a potentially useful adjunct therapy for promoting anti-tumor immunity. However, knowledge about the immunological effects of GEM intratumorally is limited. Thus, we directly compared the impact of systemic GEM on immune cell presence and functionality in the tumor microenvironment (TME) to its effects in the periphery. We found that GEM is not myeloablative in the TME; rather, we observed sustained, significant reductions in TILs and dendritic cells-crucial components in initiating an adaptive immune response. We also performed bulk-RNA sequencing to identify immunological alterations transcriptionally induced by GEM. While we found evidence of upregulation in the interferon-gamma (IFN-γ) response pathway, we determined that GEM-mediated growth control is not dependent on IFN-γ. Overall, our findings yield new insights into the tissue- and temporal-dependent immune ablative effects of GEM, contrasting the paradigm that this therapy is specifically myeloablative.

Tumor blood vessels are highly leaky in structure and have poor blood perfusion, which hampers infiltration and function of CD8T cells within tumor. Normalizing tumor vessels is thus thought to be important in promoting the flux of immune T cells and enhancing ant-tumor immunity. However, how tumor vasculature is normalized is poorly understood. Metformin (Met) combined with ant-PD-1 therapy is known to stimulate proliferation of and to produce large amounts of IFNγ from tumor-infiltrating CD8T lymphocytes (CD8TILs). We found that the combination therapy promotes the pericyte coverage of tumor vascular endothelial cells (ECs) to improve blood perfusion and that it suppresses the hyperpermeability through the increase of VE-cadherin. Peripheral node addressin(PNAd) and vascular cell adhesion molecule (VCAM)-1, both implicated to promote tumor infiltration of CD8T cells, were also increased. Importantly, tumor vessel normalization, characterized as the reduced 70-kDa dextran leakage and the enhancement of VE-cadherin and VCAM-1, were canceled by anti-CD8 Ab or anti-IFNγ Ab injection to mice. The increased CD8TILs were also abrogated by anti-IFNγ Ab injection. In vascular ECs, flow cytometry analysis revealed that pSTAT1 expression was found to be associated with VE-cadherin expression. Moreover, in vitro treatment with Met and IFNγ enhanced VE-cadherin and VCAM-1 on human umbilical vein endothelial cells (HUVECs). The Kaplan-Meier method revealed a correlation of VE-cadherin or VCAM-1 levels with overall survival in patients treated with immune checkpoint inhibitors. These data indicate that IFNγ-mediated cross talk of CD8TILs with tumor vessels is important for creating a better tumor microenvironment and maintaining sustained antitumor immunity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Tumor-infiltrating regulatory T cells (TI-Tregs) elicit immunosuppressive effects in the tumor microenvironment (TME) leading to accelerated tumor growth and resistance to immunotherapies against solid tumors. Here, we demonstrate that poly-(ADP-ribose)-polymerase-11 (PARP11) is an essential regulator of immunosuppressive activities of TI-Tregs. Expression of PARP11 correlates with TI-Treg cell numbers and poor responses to immune checkpoint blockade (ICB) in human patients with cancer. Tumor-derived factors including adenosine and prostaglandin E2 induce PARP11 in TI-Tregs. Knockout of PARP11 in the cells of the TME or treatment of tumor-bearing mice with selective PARP11 inhibitor ITK7 inactivates TI-Tregs and reinvigorates anti-tumor immune responses. Accordingly, ITK7 decelerates tumor growth and significantly increases the efficacy of anti-tumor immunotherapies including ICB and adoptive transfer of chimeric antigen receptor (CAR) T cells. These results characterize PARP11 as a key driver of TI-Treg activities and a major regulator of immunosuppressive TME and argue for targeting PARP11 to augment anti-cancer immunotherapies., Competing Interests: Declaration of interests M.S.C. is an inventor on an OHSU patent related to the general use of the ITK7 compound. M.S.C., R.B., and S.Y.F. are co-inventors on a pending patent from OHSU/University of Pennsylvania related to ITK7 use., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The treatment of melanoma, the deadliest form of skin cancer, has greatly benefited from immunotherapy. However, many patients do not show a durable response, which is only partially explained by known resistance mechanisms., Methods: We performed single-cell RNA sequencing of tumor immune infiltrates and matched peripheral blood mononuclear cells of 22 checkpoint inhibitor (CPI)-naive stage III-IV metastatic melanoma patients. After sample collection, the same patients received CPI treatment, and their response was assessed., Findings: CPI responders showed high levels of classical monocytes in peripheral blood, which preferentially transitioned toward CXCL9-expressing macrophages in tumors. Trajectories of tumor-infiltrating CD8 + T cells diverged at the level of effector memory/stem-like T cells, with non-responder cells progressing into a state characterized by cellular stress and apoptosis-related gene expression. Consistently, predicted non-responder-enriched myeloid-T/natural killer cell interactions were primarily immunosuppressive, while responder-enriched interactions were supportive of T cell priming and effector function., Conclusions: Our study illustrates that the tumor immune microenvironment prior to CPI treatment can be indicative of response. In perspective, modulating the myeloid and/or effector cell compartment by altering the described cell interactions and transitions could improve immunotherapy response., Funding: This research was funded by Roche Pharma Research and Early Development., Competing Interests: Declaration of interests R.S., S. Hoves, M.B., T.H., S.D., E.Y., F.L., S. Herter, P.U., A.I., J.S., J.S.-P., T.K.-T., I.G.d.M., and P.C.S. are employed by and hold F. Hoffmann-La Roche Ltd. company stock. P.U. and M.B. disclose ownership of F. Hoffmann-La Roche Ltd. patents. R.D. has intermittent, project-focused consulting and/or advisory relationships with Novartis, Merck Sharp & Dohme (MSD), Bristol-Myers Squibb (BMS), Roche, Amgen, Takeda, Pierre Fabre, Sun Pharma, Sanofi, Catalym, Second Genome, Regeneron, Alligator, T3 Pharma, MaxiVAX SA, Pfizer, and touchIME outside of the submitted work. M.P.L. receives project-specific research support outside of the scope of this work from Roche, Novartis, Molecular Partners, and Oncobit., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite its long history of toxicity and limited efficacy, IL2 has re-entered the clinic as a companion to the recently FDA-approved tumor infiltrating lymphocyte therapy. In back-to-back articles, Moynihan and Kaptein introduce a new fusion protein that delivers a biased IL2 mutein to CD8 T cells. See related article by Moynihan et al., p. 1206 (6). See related article by Kaptein et al., p. 1226 (7)., (©2024 American Association for Cancer Research.)

Objective: To determine the survival benefit and immunomodulatory effects of cimetidine pre-, peri- or post-operatively in patients with colorectal cancer (CRC)., Methods: A systematic review was conducted using PubMed and Cochrane Library to retrieve randomized control trials (RCTs) that investigated the effects of cimetidine on survival and immunomodulation via improvement in tumor infiltrating lymphocytes (TILs) and peripheral blood lymphocytes. The review was carried out in accordance with the extended Preferred Reporting Items for Systematic Reviews and Meta-analyses., Results: Four studies with the total of 267 patients were included in this systematic review. Treatment duration varied from 5 days to 1 year. Two studies reported a significant TIL response in the resected specimens after administering cimetidine, while one RCT showed an escalation of CD 3 , CD 4 and CD 57 lymphocytes in peripheral blood compared to the baseline following cimetidine treatment ( p  < 0.01). Of the three trials that examined the effects of cimetidine on survival, only two studies revealed significant survival benefit while the remaining study only showed a trend towards survival benefit., Conclusion: Repurposing of existing drugs like cimetidine has a potential to offer a survival benefit by acting as an immunomodulatory agent in patients undergoing curative resection for CRC. However, the heterogeneity seen in current studies and the evolvement of adjunctive therapies for CRC warrant large-scale, well-designed prospective RCTs to establish the efficacy of cimetidine in CRC., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Long-term daily use of aspirin reduces incidence and mortality due to colorectal cancer (CRC). This study aimed to analyze the effect of aspirin on the tumor microenvironment, systemic immunity, and on the healthy mucosa surrounding cancer., Methods: Patients with a diagnosis of CRC operated on from 2015 to 2019 were retrospectively analyzed (METACCRE cohort). Expression of mRNA of immune surveillance-related genes (PD-L1, CD80, CD86, HLA I, and HLA II) in CRC primary cells treated with aspirin were extracted from Gene Expression Omnibus-deposited public database (GSE76583). The experiment was replicated in cell lines. The mucosal immune microenvironment of a subgroup of patients participating in the IMMUNOREACT1 (ClinicalTrials.gov NCT04915326) project was analyzed with immunohistochemistry and flow cytometry., Results: In the METACCRE Cohort, 12% of 238 patients analyzed were aspirin users. Nodal metastasis was significantly less frequent (p = .008) and tumor-infiltrating lymphocyte infiltration was higher (p = .02) among aspirin users. In the CRC primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment (p = .001). In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users (p = .027 and p = .034, respectively)., Conclusions: These data suggested that regular aspirin use may have an active role in enhancing immunosurveillance against CRC., (© 2024 American Cancer Society.)

Objectives: Antiangiogenic inhibitors have been shown to synergize with immune checkpoint blockade, but the underlying mechanisms of the synergistic response are not fully understood., Patients and Methods: We investigate the impact of VEGFR2 inhibition on tumor-infiltrating immune cells in vivo and the activity of the combination of apatinib and anti-PD-1 in synergistic mouse model of HNSCC. A patient with squamous cell carcinoma of the left tongue with cervical lymph node were received with combined induction treatment of camrelizumab and apatinib to validate the efficacy of neoadjuvant immunotherapy before surgery., Results: We found that apatinib increased the infiltration of CD8 + T cells and decreased the population of Tregs in a preclinical syngeneic mouse model. The proportions of CD8 + PD1 + T cells were significantly increased in apatinib-treated tumors. The combined treatment of apatinib and anti-PD-1 demonstrated better therapeutic benefit than each treatment alone. The patient with squamous cell carcinoma of the left tongue with cervical lymph node achieved major pathologic response (MPR) after two cycles of combined induction treatment., Conclusion: Our study demonstrated that apatinib therapy synergized with an anti-PD-1 antibody in preclinical cancer models and in patient with advanced HNSCC. These results provide a new rationale for advancing this neoadjuvant immunotherapy in large scale of clinical trials of HNSCC., (© 2023 Wiley Periodicals LLC.)

B‑cell lymphoma is difficult to cure because of its biological and clinical heterogeneity, and due to native chemoresistance. Immunotherapies that overcome cancer‑induced immune evasion have been the center of recent developments in oncology. This is emphasized by the accomplishment of various agents that disrupt programmed cell death protein 1 (PD‑1)‑mediated immune suppression in diverse tumors. However, while PD‑1 blockade has been effective in numerous malignancies, a significant proportion of cancers, including B‑cell lymphoma, show certain rates of primary resistance to these therapeutic strategies. Histone deacetylase inhibitors (HDACis) have exhibited anticancer activity though suppressing cell proliferation, inducing differentiation and triggering apoptosis. The present study aimed to explore a therapeutic strategy combining a HDACi (romidepsin) and PD‑1 blockade (BMS‑1) in B‑cell lymphoma, utilizing a constructed mouse model of B‑cell lymphoma. The IC 50 of the two inhibitors was confirmed by MTT assay, and their inhibitory effects were revealed to be dose‑ and time‑dependent. The data demonstrated that the combined treatment of romidepsin and BMS‑1 synergistically inhibited the growth of B‑cell lymphoma. Furthermore, it was revealed that romidepsin and BMS‑1 synergistically triggered apoptosis in mouse B‑cell lymphoma. The synergistic effect of these agents was capable of activating tumor‑infiltrating lymphocytes, particularly CD3 + CD4 + and CD3 + CD8 + T cells. The results of the present study underscore the potential of HDAC inhibition in conjunction with PD‑1 blockade as a novel therapeutic approach for B‑cell lymphoma, highlighting the synergistic effects of these two mechanisms in enhancing antitumor immunity.

Immunotherapy, particularly the use of immune checkpoint inhibitors (ICIs), has shown limited efficacy in treating ovarian cancer (OC), possibly due to diverse T cell infiltration patterns in the tumor microenvironment. This review explores how neoadjuvant chemotherapy (NACT) impacts the immune landscape of OC, focusing on tumor-infiltrating lymphocytes (TILs), PD-1/PD-L1 expression, and their clinical implications. A comprehensive literature search across four databases yielded nine relevant studies. These studies evaluated stromal (sTILs) and intra-epithelial (ieTILs) TILs before and after NACT. sTIL responses varied, impacting prognostic outcomes, and ieTILs increased in some patients without clear survival associations. PD-L1 expression after NACT correlated with improved overall survival (OS), and increases in granzyme B+ and PD-1 correlated with longer progression-free survival (PFS). Remarkably, reduced FoxP3+ TILs post-NACT correlated with better prognosis. NACT often increases sTIL/ieTIL and CD8+ subpopulations, but their correlation with improved PFS and OS varies. Upregulation of co-inhibitory molecules, notably PD-L1, suggests an immunosuppressive response to chemotherapy. Ongoing trials exploring neoadjuvant ICIs and chemotherapy offer promise for advancing OC treatment. Standardized measurements assessing TIL density, location, and heterogeneity are crucial for addressing genetic complexity and immunological heterogeneity in OC.

Despite the important breakthroughs of immune checkpoint inhibitors in recent years, the objective response rates remain limited. Here, we synthesize programmed cell death protein-1 (PD-1) antibody-iRGD cyclic peptide conjugate (αPD-1-(iRGD) 2 ) through glycoengineering methods. In addition to enhancing tissue penetration, αPD-1-(iRGD) 2 simultaneously engages tumor cells and PD-1 + T cells via dual targeting, thus mediating tumor-specific T cell activation and proliferation with mild effects on non-specific T cells. In multiple syngeneic mouse models, αPD-1-(iRGD) 2 effectively reduces tumor growth with satisfactory biosafety. Moreover, results of flow cytometry and single-cell RNA-seq reveal that αPD-1-(iRGD) 2 remodels the tumor microenvironment and expands a population of "better effector" CD8 + tumor infiltrating T cells expressing stem- and memory-associated genes, including Tcf7, Il7r, Lef1, and Bach2. Conclusively, αPD-1-(iRGD) 2 is a promising antibody conjugate therapeutic beyond antibody-drug conjugate for cancer immunotherapy., Competing Interests: Declaration of interests J.W., Y.P., Y.Y., Z.S., J.P.L., and Q.X. are together applying for a domestic patent regarding the design and synthesis of αPD-1-(iRGD)(2)., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulatory T cells (Tregs) are known to facilitate tumor progression by suppressing CD8+ T cells within the tumor microenvironment (TME), thereby also hampering the effectiveness of immune checkpoint inhibitors (ICIs). While systemic depletion of Tregs can enhance antitumor immunity, it also triggers undesirable autoimmune responses. Therefore, there is a need for therapeutic agents that selectively target Tregs within the TME without affecting systemic Tregs. In this study, as shown also by others, the chemokine (C-C motif) receptor 8 (CCR8) was found to be predominantly expressed on Tregs within the TME of both humans and mice, representing a unique target for selective depletion of tumor-residing Tregs. Based on this, we developed BAY 3375968, a novel anti-human CCR8 antibody, along with respective surrogate anti-mouse CCR8 antibodies, and demonstrated their in vitro mode-of-action through induction of potent antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities. In vivo, anti-mouse CCR8 antibodies effectively depleted Tregs within the TME primarily via ADCP, leading to increased CD8+ T cell infiltration and subsequent tumor growth inhibition across various cancer models. This monotherapeutic efficacy was significantly enhanced in combination with ICIs. Collectively, these findings suggest that CCR8 targeting represents a promising strategy for Treg depletion in cancer therapies. BAY 3375968 is currently under investigation in a Phase I clinical trial (NCT05537740)., (© 2024. The Author(s).)

Tumor-infiltrating lymphocyte (TIL) deficiency is the most conspicuous obstacle to limit the cancer immunotherapy. Immune checkpoint inhibitors (ICIs), such as anti-PD-1 antibody, have achieved great success in clinical practice. However, due to the limitation of response rates of ICIs, some patients fail to benefit from monotherapy. Thus, novel combination therapy that could improve the response rates emerges as new strategies for cancer treatment. Here, we reported that the natural product rocaglamide (RocA) increased tumor-infiltrating T cells and promoted Th17 differentiation of CD4 + TILs. Despite RocA monotherapy upregulated PD-1 expression of TILs, which was considered as the consequence of T cell activation, combining RocA with anti-PD-1 antibody significantly downregulated the expression of PD-1 and promoted proliferation of TILs. Taken together, these findings demonstrated that RocA could fuel the T cell anti-tumor immunity and revealed the remarkable potential of RocA as a therapeutic candidate when combining with the ICIs., (© 2024. The Author(s).)

Purpose: To explore the cellular cross-talk of tumor-resident mast cells (MC) in controlling the activity of cancer-associated fibroblasts (CAF) to overcome tumor microenvironment (TME) abnormalities, enhancing the efficacy of immune-checkpoint inhibitors in sarcoma., Experimental Design: We used a coculture system followed by further validation in mouse models of fibrosarcoma and osteosarcoma with or without administration of the MC stabilizer and antihistamine ketotifen. To evaluate the contribution of ketotifen in sensitizing tumors to therapy, we performed combination studies with doxorubicin chemotherapy and anti-PD-L1 (B7-H1, clone 10F.9G2) treatment. We investigated the ability of ketotifen to modulate the TME in human sarcomas in the context of a repurposed phase II clinical trial., Results: Inhibition of MC activation with ketotifen successfully suppressed CAF proliferation and stiffness of the extracellular matrix accompanied by an increase in vessel perfusion in fibrosarcoma and osteosarcoma as indicated by ultrasound shear wave elastography imaging. The improved tissue oxygenation increased the efficacy of chemoimmunotherapy, supported by enhanced T-cell infiltration and acquisition of tumor antigen-specific memory. Importantly, the effect of ketotifen in reducing tumor stiffness was further validated in sarcoma patients, highlighting its translational potential., Conclusions: Our study suggests the targeting of MCs with clinically administered drugs, such as antihistamines, as a promising approach to overcome resistance to immunotherapy in sarcomas., (©2024 The Authors; Published by the American Association for Cancer Research.)

Immune checkpoint blockade (ICB) therapies function by alleviating immunosuppression on tumor-infiltrating lymphocytes (TILs) but are often insufficient to fully reactivate these dysfunctional TILs. Although interleukin 12 (IL-12) has been used in combination with ICB to improve efficacy, this remains limited by severe toxicity associated with systemic administration of this cytokine. Here, we engineer a fusion protein composed of an anti-PD-1 antibody and a mouse low-affinity IL-12 mutant-2 (αPD1-mIL12mut2). Systemic administration of αPD1-mIL12mut2 displays robust antitumor activities with undetectable toxicity. Mechanistically, αPD1-mIL12mut2 preferentially activates tumor-infiltrating PD-1 + CD8 + T cells via high-affinity αPD-1 mediated cis-binding of low-affinity IL-12. Additionally, αPD1-mIL12mut2 treatment exerts an abscopal effect to suppress distal tumors, as well as metastasis. Collectively, αPD1-mIL12mut2 treatment induces robust systemic antitumor responses with reduced side effects., (© 2024. The Author(s).)

Background: The lack of an efficient way to screen patients who are responsive to immunotherapy challenges PD1/CTLA4-targeting cancer treatment. Immunotherapeutic efficacy cannot be clearly determined by peripheral blood analyses, tissue gene markers or CT/MR value. Here, we used a radionuclide and imaging techniques to investigate the novel dual targeted antibody cadonilimab (AK104) in PD1/CTLA4-positive cells in vivo., Methods: First, humanized PD1/CTLA4 mice were purchased from Biocytogen Pharmaceuticals (Beijing) Co., Ltd. to express hPD1/CTLA4 in T-cells. Then, mouse colon cancer MC38-hPD-L1 cell xenografts were established in humanized mice. A bispecific antibody targeting PD1/CTLA4 (AK104) was labeled with radio-nuclide iodine isotopes. Immuno-PET/CT imaging was performed using a bispecific monoclonal antibody (mAb) probe 124 I-AK104, developed in-house, to locate PD1 + /CTLA4 + tumor-infiltrating T cells and monitor their distribution in mice to evaluate the therapeutic effect., Results: The 124 I-AK104 dual-antibody was successfully constructed with ideal radiochemical characteristics, in vitro stability and specificity. The results of immuno-PET showed that 124 I-AK104 revealed strong hPD1/CTLA4-positive responses with high specificity in humanized mice. High uptake of 124 I-AK104 was observed not only at the tumor site but also in the spleen. Compared with PD1- or CTLA4-targeting mAb imaging, 124 I-AK104 imaging had excellent standard uptake values at the tumor site and higher tumor to nontumor (T/NT) ratios., Conclusions: The results demonstrated the potential of translating 124 I-AK104 into a method for screening patients who benefit from immunotherapy and the efficacy, as well as the feasibility, of this method was verified by immuno-PET imaging of humanized mice., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Ovarian cancer is the most lethal and aggressive gynecological cancer with a high recurrence rate and is often diagnosed late. In ovarian cancer, multiple metabolic enzymes of lipid metabolism are abnormally expressed, resulting in metabolism disorder. As a characteristic pathway in polyunsaturated fatty acid (PUFA) metabolism, arachidonic acid (AA) metabolism is disturbed in ovarian cancer. Therefore, we established a 10-gene signature model to evaluate the prognostic risk of PUFA-related genes. This 10-gene signature has strong robustness and can play a stable predictive role in datasets of various platforms (TCGA, ICGC, and GSE17260). The high association between the risk subgroups and clinical characteristics indicated a good performance of the model. Our data further indicated that the high expression of LTA4H was positively correlated with poor prognosis in ovarian cancer. Deficiency of LTA4H enhanced sensitivity to Cisplatin and modified the characteristics of immune cell infiltration in ovarian cancer. Additionally, our results indicate that CCL5 was involved in the aberrant metabolism of the AA/LTA4H axis, which contributes to the reduction of tumor-infiltrating CD8 + T cells and immune escape in ovarian cancer. These findings provide new insights into the prognosis and potential target of LTA4H/CCL5 in treating ovarian cancer., (© 2024. Science China Press.)

Objective: Up-regulating programmed cell death ligand-1(PD-L1) expressed on tumor cells and tumor-infiltrating myeloid cells interacting with up-regulated programmed cell death-1 (PD-1) expressed on tumor-infiltrating lymphoid cells greatly hinder their tumor-inhibiting effect. It is necessary to explore the deep mechanism of this negative effect, so as to find the potential methods to improve the immunotherapy efficiency., Methods and Results: In this study, we found that the PD-1 expression in lung cancer-infiltrating type II innate lymphoid cells (ILC2s) was highly up-regulated, which greatly restrained the activation and function of ILC2s. Furthermore, anti-PD-1 could restore the inhibition and effective cytokine secretion of ILC2s when co-cultured with tumor cells. In vivo studies proved that anti-PD-1 treatment promoted the activation of tumor-infiltrating ILC2s and inhibited the tumor growth of LLC-bearing nude mice., Discussion: Our studies demonstrate a new PD-1/PD-L1 axis regulating mechanism on innate immune cells, which provide a useful direction to ILC2s-based immunotherapy to cancer diseases.

High-grade osteosarcoma, a primary malignant bone tumour, is experiencing a global increase in reported incidence with varied prevalence. Despite advances in management, which include surgery and neoadjuvant chemotherapy often an unsatisfactory outcome is found due to poor or heterogeneous response to chemotherapy. Our study delved into chemotherapy responses in osteosarcoma patients and associated molecular expressions, focusing on CD95 receptor (CD95R), interferon (IFN)-γ, catalase, heat-shock protein (Hsp)70, and vascular endothelial growth factor (VEGF). Employing immunohistochemistry and Huvos grading of post-chemo specimens, we analysed formalin-fixed paraffin-embedded (FFPE) osteosarcoma tissue of resected post-chemotherapy specimens from Dr. Soetomo General Academic Hospital in Surabaya, Indonesia (DSGAH), spanning from 2016 to 2020. Results revealed varied responses (poor 40.38%, moderate 48.08%, good 11.54%) and distinct patterns in CD95R, IFN-γ, catalase, Hsp70, and VEGF expression. Significant differences among response groups were observed in CD95R and IFN-γ expression in tumour-infiltrating lymphocytes. The trend of diminishing CD95R expression from poor to good responses, accompanied by an increase in IFN-γ, implied a reduction in the count of viable osteosarcoma cells with the progression of Huvos grading. Catalase expression in osteosarcoma cells was consistently elevated in the poor response group, while Hsp70 expression was highest. VEGF expression in macrophages was significantly higher in the good response group. In conclusion, this study enhances our understanding of immune-chemotherapy interactions in osteosarcoma and identifies potential biomarkers for targeted interventions., (© 2024. The Author(s).)

Background & Aims: Although the presence of tertiary lymphoid structures (TLS) correlates with positive responses to immunotherapy in many solid malignancies, the mechanism by which TLS enhances antitumor immunity is not well understood. The present study aimed to investigate the underlying cross talk circuits between B cells and tissue-resident memory T (Trm) cells within the TLS and to understand their role in the context of immunotherapy., Methods: Immunostaining and H&E staining of TLS and chemokine (C-X-C motif) ligand 13 (CXCL13) + cluster of differentiation (CD)103 + CD8 + Trm cells were performed on tumor sections from patients with gastric cancer (GC). The mechanism of communication between B cells and CXCL13 + CD103 + CD8 + Trm cells was determined in vitro and in vivo. The effect of CXCL13 + CD103 + CD8 + Trm cells in suppressing tumor growth was evaluated through anti-programmed cell death protein (PD)-1 therapy., Results: The presence of TLS and CXCL13 + CD103 + CD8 + Trm cells in tumor tissues favored a superior response to anti-PD-1 therapy in patients with GC. Additionally, our research identified that activated B cells enhanced CXCL13 and granzyme B secretion by CD103 + CD8 + Trm cells. Mechanistically, B cells facilitated the glycolysis of CD103 + CD8 + Trm cells through the lymphotoxin-α/tumor necrosis factor receptor 2 (TNFR2) axis, and the mechanistic target of rapamycin signaling pathway played a critical role in CD103 + CD8 + Trm cells glycolysis during this process. Moreover, the presence of TLS and CXCL13 + CD103 + CD8 + Trm cells correlated with potent responsiveness to anti-PD-1 therapy in a TNFR2-dependent manner., Conclusions: This study further reveals a crucial role for cellular communication between TLS-associated B cell and CXCL13 + CD103 + CD8 + Trm cells in antitumor immunity, providing valuable insights into the potential use of the lymphotoxin-α/TNFR2 axis within CXCL13 + CD103 + CD8 + Trm cells for advancing immunotherapy strategies in GC., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Non-small cell lung cancer (NSCLC) is a leading cause of mortality worldwide and requires effective treatment strategies. Recently, the development of a novel multiple-target tyrosine kinase inhibitor, anlotinib, has drawn increasing attention, especially it shows advantages when combined with PD-1/PD-L1 blockade. However, the mechanism by which anlotinib improves immunotherapy and remodeling of the tumor microenvironment remains unclear. In this study, we found that anlotinib combined with PD-1 blockade significantly inhibited tumor growth and reduced tumor weight in a lung cancer xenograft model compared to any single treatment. Both immunofluorescence and flow cytometry analyses revealed that anlotinib induced a CD8 + T cell dominated tumor microenvironment, which might account for its improved role in immunotherapy. Further investigations showed that CCL5-mediated CD8 + T cell recruitment plays a critical role in anlotinib and PD-1 blockade strategies. The depletion of CD8 + T cells abrogated this process. In conclusion, our findings showed that the combination of anlotinib and PD-1 blockade produced promising effects in the treatment of lung cancer, and that the induction of CCL5-mediced CD8 + T cell recruitment by anlotinib provided a novel mechanism of action., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Identifying tumor-relevant T cell subsets in the peripheral blood (PB) has become a potential strategy for cancer treatment. However, the subset of PB that could be used to treat cancer remains poorly defined. Here, we found that the CX3CR1 + T cell subset in the blood of patients with lung cancer exhibited effector properties and had a higher TCR matching ratio with tumor-infiltrating lymphocytes (TILs) compared to CX3CR1 - T cells, as determined by paired single-cell RNA and TCR sequencing. Meanwhile, the anti-tumor activities, effector cytokine production, and mitochondrial function were enhanced in CX3CR1 + T cells both in vitro and in vivo . However, in the co-culture system of H322 cells with T cells, the percentages of apoptotic cells and Fas were substantially higher in CX3CR1 + T cells than those in CX3CR1 - T cells. Fas-mediated apoptosis was rescued by treatment with an anti-PD-1 antibody. Accordingly, the combination of adoptive transfer of CX3CR1 + T cells and anti-PD-1 treatment considerably decreased Fas expression and improved the survival of lung xenograft mice. Moreover, an increased frequency of CX3CR1 + T cells in the PB correlated with a better response and prolonged survival of patients with lung cancer who received anti-PD-1 therapy. These findings indicate the promising potential of adoptive transfer of peripheral CX3CR1 + T cells as an individual cancer immunotherapy., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Immune therapy is the new frontier of cancer treatment. Therapeutic radiation is a known inducer of immune response and can be limited by immunosuppressive mediators including cyclooxygenase-2 (COX2) that is highly expressed in aggressive triple negative breast cancer (TNBC). A clinical cohort of TNBC tumors revealed poor radiation therapeutic efficacy in tumors expressing high COX2. Herein, we show that radiation combined with adjuvant NSAID (indomethacin) treatment provides a powerful combination to reduce both primary tumor growth and lung metastasis in aggressive 4T1 TNBC tumors, which occurs in part through increased antitumor immune response. Spatial immunological changes including augmented lymphoid infiltration into the tumor epithelium and locally increased cGAS/STING1 and type I IFN gene expression were observed in radiation-indomethacin-treated 4T1 tumors. Thus, radiation and adjuvant NSAID treatment shifts "immune desert phenotypes" toward antitumor M1/TH1 immune mediators in these immunologically challenging tumors. Importantly, radiation-indomethacin combination treatment improved local control of the primary lesion, reduced metastatic burden, and increased median survival when compared with radiation treatment alone. These results show that clinically available NSAIDs can improve radiation therapeutic efficacy through increased antitumor immune response and augmented local generation of cGAS/STING1 and type I IFNs.

Bispecific T cell engagers (TCEs) show promising clinical efficacy in blood tumors, but their application to solid tumors remains challenging. Here, we show that Fc-fused IL-7 (rhIL-7-hyFc) changes the intratumoral CD8 T cell landscape, enhancing the efficacy of TCE immunotherapy. rhIL-7-hyFc induces a dramatic increase in CD8 tumor-infiltrating lymphocytes (TILs) in various solid tumors, but the majority of these cells are PD-1-negative tumor non-responsive bystander T cells. However, they are non-exhausted and central memory-phenotype CD8 T cells with high T cell receptor (TCR)-recall capacity that can be triggered by tumor antigen-specific TCEs to acquire tumoricidal activity. Single-cell transcriptome analysis reveals that rhIL-7-hyFc-induced bystander CD8 TILs transform into cycling transitional T cells by TCE redirection with decreased memory markers and increased cytotoxic molecules. Notably, TCE treatment has no major effect on tumor-reactive CD8 TILs. Our results suggest that rhIL-7-hyFc treatment promotes the antitumor efficacy of TCE immunotherapy by increasing TCE-sensitive bystander CD8 TILs in solid tumors., Competing Interests: Declaration of interests M.L., S.-K.I., and D.C. are employees of the Research Institute of NeoImmuneTech, Inc., which supports the supply of rhIL-7-hyFc and its formulation buffer., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Tumor-infiltrating lymphocyte (TIL) therapy has been restricted by intensive lymphodepletion and high-dose intravenous interleukin-2 (IL-2) administration. To address these limitations, we conducted preclinical and clinical studies to evaluate the safety, antitumor activity, and pharmacokinetics of an innovative modified regimen in patients with advanced gynecologic cancer., Methods: Patient-derived xenografts (PDX) were established from a local recurrent cervical cancer patient. TILs were expanded ex vivo from minced tumors without feeder cells in the modified TIL therapy regimen. Patients underwent low-dose cyclophosphamide lymphodepletion followed by TIL infusion without intravenous IL-2. The primary endpoint was safety; the secondary endpoints included objective response rate, duration of response, and T cell persistence., Results: In matched patient-derived xenografts (PDX) models, homologous TILs efficiently reduced tumor size (p < 0.0001) and underwent IL-2 absence in vivo. In the clinical section, all enrolled patients received TIL infusion using a modified TIL therapy regimen successfully with a manageable safety profile. Five (36%, 95% CI 16.3-61.2) out of 14 evaluable patients experienced objective responses, and three complete responses were ongoing at 19.5, 15.4, and 5.2 months, respectively. Responders had longer overall survival (OS) than non-responders (p = 0.036). Infused TILs showed continuous proliferation and long-term persistence in all patients and showed greater proliferation in responders which was indicated by the Morisita overlap index (MOI) of TCR clonotypes between infused TILs and peripheral T cells on day 14 (p = 0.004) and day 30 (p = 0.004). Higher alteration of the CD8 + /CD4 + ratio on day 14 indicated a longer OS (p = 0.010)., Conclusions: Our modified TIL therapy regimen demonstrated manageable safety, and TILs could survive and proliferate without IL-2 intravenous administration, showing potent efficacy in patients with advanced gynecologic cancer., Trial Registration: NCT04766320, Jan 04, 2021., (© 2024. The Author(s).)

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate contributions to purine nucleotides from salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic or lymph node T cells) synthesize purines de novo. Shortage of 1C units for T cell purine synthesis is accordingly a potential bottleneck for anti-tumor immunity. Supplementing 1C units by infusing formate drives formate assimilation into purines in tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling kinetic control of formate production. Safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade in MC38 tumors, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation., Competing Interests: Declaration of interests J.D.R. is a paid adviser and/or stockholder in Colorado Research Partners, L.E.A.F. Pharmaceuticals, Faeth Therapeutics, and Empress Therapeutics; a paid consultant of Pfizer; a founder and stockholder in Marea Therapeutics; a founder, director, and stockholder of Farber Partners, Raze Therapeutics, and Sofro Pharmaceuticals. J.D.R. and K.O. are inventors of patent applications related to metabolic supplements that enhance immunotherapy., (Copyright © 2024. Published by Elsevier Ltd.)

Purpose: Clinical implications of neoadjuvant immunotherapy in patients with locally advanced but resectable head and neck squamous cell carcinoma (HNSCC) remain largely unexplored., Patients and Methods: Patients with resectable HNSCC were randomized to receive a single dose of preoperative durvalumab (D) with or without tremelimumab (T) before resection, followed by postoperative (chemo)radiotherapy based on multidisciplinary discretion and 1-year D treatment. Artificial intelligence (AI)-powered spatial distribution analysis of tumor-infiltrating lymphocytes and high-dimensional profiling of circulating immune cells tracked dynamic intratumoral and systemic immune responses., Results: Of the 48 patients enrolled (D, 24 patients; D+T, 24 patients), 45 underwent surgical resection per protocol (D, 21 patients; D+T, 24 patients). D±T had a favorable safety profile and did not delay surgery. Distant recurrence-free survival (DRFS) was significantly better in patients treated with D+T than in those treated with D monotherapy. AI-powered whole-slide image analysis demonstrated that D+T significantly reshaped the tumor microenvironment toward immune-inflamed phenotypes, in contrast with the D monotherapy or cytotoxic chemotherapy. High-dimensional profiling of circulating immune cells revealed a significant expansion of T-cell subsets characterized by proliferation and activation in response to D+T therapy, which was rare following D monotherapy. Importantly, expansion of specific clusters in CD8+ T cells and non-regulatory CD4+ T cells with activation and exhaustion programs was associated with prolonged DRFS in patients treated with D+T., Conclusions: Preoperative D±T is feasible and may benefit patients with resectable HNSCC. Distinct changes in the tumor microenvironment and circulating immune cells were induced by each treatment regimen, warranting further investigation., (©2024 American Association for Cancer Research.)

Background: Staphylococcal enterotoxin C2 (SEC2) is used as an immunotherapeutic drug in China. However, SEC2 are limited due to its immunosuppressive and toxic effects. A SEC2 2M-118 (H118A/T20L/G22E) mutant generated by site-directed mutagenesis was studied to elucidate the underlying antitumor mechanism., Methods: The effects of 2M-118 on mouse fibrosarcoma (Meth-A) cells and cytokine responses were tested in vitro using a transwell assay and ELISA, respectively. 2M-118 effect on immune function in tumor-bearing mice was tested. Cytokine levels and antitumor responses were measured using ELISA and flow cytometry, respectively. TUNEL staining and immunohistochemistry were employed to detect the tumor apoptosis and CD4 + and CD8 + tumor infiltrating lymphocytes (TILs) in tumor tissue., Results: 2M-118 demonstrated the growth inhibition on tumor cells, increase of cytokines production (IL-2, IFN-γ, and TNF-α) and splenocyte proliferation in vitro. 2M-118 effectively inhibited tumor development and increased lymphocytes and cytokines in a tumor-bearing mouse model. Additionally, 2M-118 regulated the tumormicroenvironment by reducing the number of myeloid-derived suppressor cells (MDSCs), increasing the number of TILs, and inducing tumorcell apoptosis., Conclusion: 2M-118 promotes immune function and enhances antitumor response. This indicates that 2M-118 could potentially be developed as a novel anti-tumor drug with-highefficiencyandlowtoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Head and neck cancer (HNC) is prevalent worldwide, and treatment options are limited. Momordicine-I (M-I), a natural component from bitter melon, shows antitumor activity against these cancers, but its mechanism of action, especially in the tumor microenvironment (TME), remains unclear. In this study, we establish that M-I reduces HNC tumor growth in two different immunocompetent mouse models using MOC2 and SCC VII cells. We demonstrate that the anticancer activity results from modulating several molecules in the monocyte/macrophage clusters in CD45+ populations in MOC2 tumors by single-cell RNA sequencing. Tumor-associated macrophages (TAM) often pose a barrier to antitumor effects, but following M-I treatment, we observe a significant reduction in the expression of Sfln4, a myeloid cell differentiation factor, and Cxcl3, a neutrophil chemoattractant, in the monocyte/macrophage populations. We further find that the macrophages must be in close contact with the tumor cells to inhibit Sfln4 and Cxcl3, suggesting that these TAMs are impacted by M-I treatment. Coculturing macrophages with tumor cells shows inhibition of Agr1 expression following M-I treatment, which is indicative of switching from M2 to M1 phenotype. Furthermore, the total B-cell population in M-I-treated tumors is significantly lower, whereas spleen cells also show similar results when cocultured with MOC2 cells. M-I treatment also inhibits PD1, PD-L1, and FoxP3 expression in tumors. Collectively, these results uncover the potential mechanism of M-I by modulating immune cells, and this new insight can help to develop M-I as a promising candidate to treat HNCs, either alone or as adjuvant therapy., (©2024 American Association for Cancer Research.)

There have been many recent advances in melanoma therapy. While 50% of melanomas have a BRAF mutation and are a target for BRAF inhibitors, the remaining 50% are BRAF wild-type. Immune checkpoint inhibitors targeting PD-1, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and lymphocyte activated gene-3 (Lag-3) are all approved for the treatment of patients with advanced BRAF wild-type melanoma; however, treatment of this patient population following initial immune checkpoint blockade is a current therapeutic challenge given the lack of other efficacious options. Here, we briefly review available US FDA-approved therapies for BRAF wild-type melanoma and focus on developing treatment avenues for this heterogeneous group of patients. We review the basics of genomic features of both BRAF mutant and BRAF wild-type melanoma as well as efforts underway to develop new targeted therapies involving the mitogen-activated protein kinase (MAPK) pathway for patients with BRAF wild-type tumors. We then focus on novel immunotherapies, including developing checkpoint inhibitors and agonists, cytokine therapies, oncolytic viruses and tumor-infiltrating lymphocytes, all of which represent potential therapeutic avenues for patients with BRAF wild-type melanoma who progress on currently approved immune checkpoint inhibitors., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Immune checkpoint therapy (ICT) causes durable tumour responses in a subgroup of patients, but it is not well known how T cell receptor beta (TCRβ) repertoire dynamics contribute to the therapeutic response. Using murine models that exclude variation in host genetics, environmental factors and tumour mutation burden, limiting variation between animals to naturally diverse TCRβ repertoires, we applied TCRseq, single cell RNAseq and flow cytometry to study TCRβ repertoire dynamics in ICT responders and non-responders. Increased oligoclonal expansion of TCRβ clonotypes was observed in responding tumours. Machine learning identified TCRβ CDR3 signatures unique to each tumour model, and signatures associated with ICT response at various timepoints before or during ICT. Clonally expanded CD8+ T cells in responding tumours post ICT displayed effector T cell gene signatures and phenotype. An early burst of clonal expansion during ICT is associated with response, and we report unique dynamics in TCRβ signatures associated with ICT response., Competing Interests: LB is employed by the company JJP Biologics. WJL received research funding from Douglas Pharmaceuticals, AstraZeneca, ENA therapeutics, consultancy for Douglas Pharmaceuticals and MSD. AN is on the advisory board of Boehringer Ingelheim, Bayer, Roche, BMS; and received research funding from AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The insufficient abundance and weak activity of tumour-infiltrating lymphocytes (TILs) are two important reasons for the poor efficacy of PD-1 inhibitors in hepatocellular carcinoma (HCC) treatment. The combined administration of tanshinone II A (TSA) and astragaloside IV (As) can up-regulate the abundance and activity of TILs by normalising tumour blood vessels and reducing the levels of immunosuppressive factors respectively. For enhancing the efficacy of PD-1 antibody, a magnetic metal-organic framework (MOF) with a homologous tumour cell membrane (Hm) coating (Hm@TSA/As-MOF) is established to co-deliver TSA&As into the HCC microenvironment. Hm@TSA/As-MOF is a spherical nanoparticle and has a high total drug-loading capacity of 16.13 wt%. The Hm coating and magnetic responsiveness of Hm@TSA/As-MOF provide a homologous-magnetic dual-targeting, which enable Hm@TSA/As-MOF to counteract the interference posed by ascites tumour cells and enhance the precision of targeting solid tumours. Hm coating also enable Hm@TSA/As-MOF to evade immune clearance by macrophages. The release of TSA&As from Hm@TSA/As-MOF can be accelerated by HCC microenvironment, thereby up-regulating the abundance and activity of TILs to synergistic PD-1 antibody against HCC. This study presents a nanoplatform to improve the efficacy of PD-1 inhibitors in HCC, providing a novel approach for anti-tumour immunotherapy in clinical practice., (© 2024. The Author(s).)

Background: Immune-checkpoint inhibitors (ICIs) against programmed death (PD)-1/PD-L1 pathway immunotherapy have been demonstrated to be effective in only a subset of patients with cancer, while the rest may exhibit low response or may develop drug resistance after initially responding. Previous studies have indicated that extensive collagen-rich stroma secreted by cancer-associated fibroblasts (CAFs) within the tumor microenvironment is one of the key obstructions of the immunotherapy for some tumors by decreasing the infiltrating cytotoxic T cells. However, there is still a lack of effective therapeutic strategies to control the extracellular matrix by targeting CAFs., Methods: The enhanced uptake of IR-780 by CAFs was assessed by using in vivo or ex vivo nearinfrared fluorescence imaging, confocal NIR fluorescent imaging, and CAFs isolation testing. The fibrotic phenotype down-regulation effects and in vitro CAFs killing effect of IR-780 were tested by qPCR, western blot, and flow cytometry. The in vivo therapeutic enhancement of anti-PD-L1 by IR-780 was evaluated on EMT6 and MC38 subcutaneous xenograft mice models., Results: IR-780 has been demonstrated to be preferentially taken up by CAFs and accumulate in the mitochondria. Further results identified low-dose IR-780 to downregulate the fibrotic phenotype, while high-dose IR-780 could directly kill both CAFs and EMT6 cells in vitro. Moreover, IR-780 significantly inhibited extracellular matrix (ECM) protein deposition in the peri-tumoral stroma on subcutaneous EMT6 and MC38 xenografts, which increased the proportion of tumor-infiltrating lymphocytes (TILs) in the deep tumor and further promoted anti-PD-L1 therapeutic efficacy., Conclusion: This work provides a unique strategy for the inhibition of ECM protein deposition in the tumor microenvironment by targeted regulating of CAFs, which destroys the T cell barrier and further promotes tumor response to PD-L1 monoclonal antibody. IR-780 has been proposed as a potential therapeutic small-molecule adjuvant to promote the effect of immunotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Myeloid cells are known to suppress antitumour immunity 1 . However, the molecular drivers of immunosuppressive myeloid cell states are not well defined. Here we used single-cell RNA sequencing of human and mouse non-small cell lung cancer (NSCLC) lesions, and found that in both species the type 2 cytokine interleukin-4 (IL-4) was predicted to be the primary driver of the tumour-infiltrating monocyte-derived macrophage phenotype. Using a panel of conditional knockout mice, we found that only deletion of the IL-4 receptor IL-4Rα in early myeloid progenitors in bone marrow reduced tumour burden, whereas deletion of IL-4Rα in downstream mature myeloid cells had no effect. Mechanistically, IL-4 derived from bone marrow basophils and eosinophils acted on granulocyte-monocyte progenitors to transcriptionally programme the development of immunosuppressive tumour-promoting myeloid cells. Consequentially, depletion of basophils profoundly reduced tumour burden and normalized myelopoiesis. We subsequently initiated a clinical trial of the IL-4Rα blocking antibody dupilumab 2-5 given in conjunction with PD-1/PD-L1 checkpoint blockade in patients with relapsed or refractory NSCLC who had progressed on PD-1/PD-L1 blockade alone (ClinicalTrials.gov identifier NCT05013450 ). Dupilumab supplementation reduced circulating monocytes, expanded tumour-infiltrating CD8 T cells, and in one out of six patients, drove a near-complete clinical response two months after treatment. Our study defines a central role for IL-4 in controlling immunosuppressive myelopoiesis in cancer, identifies a novel combination therapy for immune checkpoint blockade in humans, and highlights cancer as a systemic malady that requires therapeutic strategies beyond the primary disease site., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Since T cell exclusion contributes to tumor immune evasion and immunotherapy resistance, how to improve T cell infiltration into solid tumors becomes an urgent challenge. Methods: We employed deep learning to profile the tumor immune microenvironment (TIME) in triple negative breast cancer (TNBC) samples from TCGA datasets and noticed that fibroblast growth factor receptor (FGFR) signaling pathways were enriched in the immune-excluded phenotype of TNBC. Erdafitinib, a selective FGFR inhibitor, was then used to investigate the effect of FGFR blockade on TIME landscape of TNBC syngeneic mouse models by flow cytometry, mass cytometry (CyTOF) and RNA sequencing. Cell Counting Kit-8 (CCK-8) assay and transwell migration assay were carried out to detect the effect of FGFR blockade on cell proliferation and migration, respectively. Cytokine array, western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) were employed to investigate the potential mechanism by which FGFR inhibition enhanced T cell infiltration. Results: Blocking FGFR pathway by Erdafitinib markedly suppressed tumor growth with increased T cell infiltration in immunocompetent mouse models of TNBC. Mechanistically, FGFR blockade inhibited cancer-associated fibroblasts (CAFs) proliferation, migration and secretion of vascular cell adhesion molecule 1 (VCAM-1) by down-regulating MAPK/ERK pathway in CAFs, thus promoting T cell infiltration by breaking physical and chemical barriers built by CAFs in TIME. Furthermore, we observed that FGFR inhibition combined with immune checkpoint blockade therapy (ICT) greatly improved the therapeutic response of TNBC tumor models. Conclusions: FGFR blockade enhanced ICT response by turning immune "cold" tumor into "hot" tumor, providing remarkable implications of FGFR inhibitors as adjuvant agents for combinatorial immunotherapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The challenge to improve the clinical efficacy and enlarge the population that benefits from immune checkpoint inhibitors (ICIs) for non-small-cell lung cancer (NSCLC) is significant. Based on whole-exosome sequencing analysis of biopsies from NSCLC patients before anti-programmed cell death protein-2 (PD-1) treatment, we identified NLRP4 mutations in the responders with a longer progression-free survival (PFS). Knockdown of NLRP4 in mouse Lewis lung cancer cell line enhanced interferon (IFN)-α/β production through the cGAS-STING-IRF3/IRF7 axis and promoted the accumulation of intratumoral CD8 + T cells, leading to tumor growth retardation in vivo and a synergistic effect with anti-PD-ligand 1 therapy. This was consistent with clinical observations that more tumor-infiltrating CD8 + T cells and elevated peripheral IFN-α before receiving nivolumab treatment were associated with a longer PFS in NSCLC patients. Our study highlights the roles of tumor-intrinsic NLRP4 in remodeling the immune contextures in the tumor microenvironment, making regional type I IFN beneficial for ICI treatment., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre- and post normalization with CytofIn demonstrates effective batch correction while recapitulating the gold-standard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to integrate public datasets without necessitating identical control samples or bead standards for fast and robust analysis using CytofIn., (© 2022. The Author(s).)

Background: Radioimmunotherapy has become one of the most promising strategies for cancer treatment. Preclinical and clinical studies have demonstrated that antiangiogenic therapy can improve the efficacy of immunotherapy and sensitize radiotherapy through a variety of mechanisms. However, it is undefined whether angiogenesis inhibitors can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of anlotinib (AL3818) on the combination of radiotherapy and immune checkpoint inhibitors in Lewis lung carcinoma mouse., Methods: C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control. Immune response and immunophenotyping including the quantification and activation were determined by flow cytometry, multiplex immunofluorescence, and multiplex immunoassay., Results: Triple therapy (radiotherapy combined with anti-PD-L1 and anlotinib) increased tumor-infiltrating lymphocytes and reversed the immunosuppressive effect of radiation on the tumor microenvironment in mouse model. Compared with radioimmunotherapy, the addition of anlotinib also boosted the infiltration of CD8 + T cells and M1 cells and caused a decrease in the number of MDSCs and M2 cells in mice. The levels of IFN-gamma and IL-18 were the highest in the triple therapy group, while the levels of IL-23, IL-13, IL-1 beta, IL-2, IL-6, IL-10, and Arg-1 were significantly reduced. NF- κ B, MAPK, and AKT pathways were downregulated in triple therapy compared with radioimmunotherapy. Thus, the tumor immune microenvironment was significantly improved. As a consequence, triple therapy displayed greater benefit in antitumor efficacy., Conclusion: Our findings indicate that anlotinib might be a potential synergistic treatment for radioimmunotherapy to achieve better antitumor efficacy in NSCLC patients by potentiating the tumor immune microenvironment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Meng Yuan et al.)

PD-1 blockade exerts clinical efficacy against various types of cancer by reinvigorating T cells that directly attack tumor cells (tumor-specific T cells) in the tumor microenvironment (TME), and tumor-infiltrating lymphocytes (TILs) also comprise nonspecific bystander T cells. Here, using single-cell sequencing, we show that TILs include skewed T cell clonotypes, which are characterized by exhaustion (T ex ) or nonexhaustion signatures (T non-ex ). Among skewed clonotypes, those in the T ex , but not those in the T non-ex , cluster respond to autologous tumor cell lines. After PD-1 blockade, non-preexisting tumor-specific clonotypes in the T ex cluster appear in the TME. Tumor-draining lymph nodes (TDLNs) without metastasis harbor a considerable number of such clonotypes, whereas these clonotypes are rarely detected in peripheral blood. We propose that tumor-infiltrating skewed T cell clonotypes with an exhausted phenotype directly attack tumor cells and that PD-1 blockade can promote infiltration of such T ex clonotypes, mainly from TDLNs., Competing Interests: Declaration of interests N.S., M.I., and K.Y. are employees of KOTAI Biotechnologies Inc. T.I. received honoraria and research grants from Ono Pharmaceutical, Bristol-Myers Squibb, and MSD outside of this study. T.H. received a research grant from Ono Pharmaceutical outside of this study. H. Mano received a research grant from Ono Pharmaceutical outside of this study. H.N. received honoraria and research funding from Ono Pharmaceutical, Chugai Pharmaceutical, MSD and Bristol-Myers Squibb, and research funding from Taiho Pharmaceutical, Daiichi-Sankyo, Kyowa Kirin, Zenyaku Kogyo, Oncolys BioPharma, Debiopharm, Asahi-Kasei, Sysmex, Fujifilm, SRL, Astellas Pharmaceutical, Sumitomo Dainippon Pharma, and BD Japan outside of this study. Y.T. received a research grant from KOTAI Biotechnologies Inc. related to this study. Y.T. also received research grants and honoraria from Ono Pharmaceutical, Bristol-Myers Squibb, and Daiichi-Sankyo, and honoraria from AstraZeneca, Chugai Pharmaceutical, and MSD outside of this study. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Radiation and bacillus Calmette-Guérin (BCG) instillations are used clinically for treatment of urothelial carcinoma, but the precise mechanisms by which they activate an immune response remain elusive. The role of the cGAS-STING pathway has been implicated in both BCG and radiation-induced immune response; however, comparison of STING pathway molecules and the immune landscape following treatment in urothelial carcinoma has not been performed. We therefore comprehensively analyzed the local immune response in the bladder tumor microenvironment following radiotherapy and BCG instillations in a well-established spontaneous murine model of urothelial carcinoma to provide insight into activation of STING-mediated immune response. Mice were exposed to the oral carcinogen, BBN, for 12 weeks prior to treatment with a single 15 Gy dose of radiation or three intravesical instillations of BCG (1 × 10 8  CFU). At sacrifice, tumors were staged by a urologic pathologist and effects of therapy on the immune microenvironment were measured using the NanoString Myeloid Innate Immunity Panel and immunohistochemistry. Clinical relevance was established by measuring immune biomarker expression of cGAS and STING on a human tissue microarray consisting of BCG-treated non-muscle-invasive urothelial carcinomas. BCG instillations in the murine model elevated STING and downstream STING-induced interferon and pro-inflammatory molecules, intratumoral M1 macrophage and T-cell accumulation, and complete tumor eradication. In contrast, radiotherapy caused no changes in STING pathway or innate immune gene expression; rather, it induced M2 macrophage accumulation and elevated FoxP3 expression characteristic of immunosuppression. In human non-muscle-invasive bladder cancer, STING protein expression was elevated at baseline in patients who responded to BCG therapy and increased further after BCG therapy. Overall, these results show that STING pathway activation plays a key role in effective BCG-induced immune response and strongly indicate that the effects of BCG on the bladder cancer immune microenvironment are more beneficial than those induced by radiation. © 2021 The Pathological Society of Great Britain and Ireland., (© 2021 The Pathological Society of Great Britain and Ireland.)

Cancer immunotherapy can boost the immune response of patients to eliminate tumor cells and suppress tumor metastasis and recurrence. However, immunotherapy resistance and the occurrence of severe immune-related adverse effects are clinical challenges that remain to be addressed. The tumor microenvironment plays a crucial role in the therapeutic efficacy of cancer immunotherapy. Injectable hydrogels have emerged as powerful drug delivery platforms offering good biocompatibility and biodegradability, minimal invasion, convenient synthesis, versatility, high drug-loading capacity, controlled drug release, and low toxicity. In this review, we summarize the application of injectable hydrogels as a unique platform for targeting the immunosuppressive tumor microenvironment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Geng, Yue, Lo, Huang and Jin.)

Checkpoint inhibitors targeting PD-(L)1 induce objective responses in 20% of patients with metastatic urothelial cancer (UC). CD8 + T cell infiltration has been proposed as a putative biomarker for response to checkpoint inhibitors. Nevertheless, data on spatial and temporal heterogeneity of tumor-infiltrating lymphocytes in advanced UC are lacking. The major aims of this study were to explore spatial heterogeneity for lymphocyte infiltration and to investigate how the immune landscape changes during the disease course. We performed multiplex immunohistochemistry to assess the density of intratumoral and stromal CD3 + , CD8 + , FoxP3 + and CD20 + immune cells in longitudinally collected samples of 49 UC patients. Within these samples, spatial heterogeneity for lymphocyte infiltration was observed. Regions the size of a 0.6 tissue microarray core (0.28 mm 2 ) provided a representative sample in 60.6 to 71.6% of cases, depending on the cell type of interest. Regions of 3.30 mm 2 , the median tumor surface area in our biopsies, were representative in 58.8 to 73.8% of cases. Immune cell densities did not significantly differ between untreated primary tumors and metachronous distant metastases. Interestingly, CD3 + , CD8 + and FoxP3 + T cell densities decreased during chemotherapy in two small cohorts of patients treated with neoadjuvant or palliative platinum-based chemotherapy. In conclusion, spatial heterogeneity in advanced UC challenges the use of immune cell infiltration in biopsies as biomarker for response prediction. Our data also suggests a decrease in tumor-infiltrating T cells during platinum-based chemotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van Wilpe, Gorris, van der Woude, Sultan, Koornstra, van der Heijden, Gerritsen, Simons, de Vries and Mehra.)

Rationale: Despite evidence suggesting that the tumor microenvironment (TME) in malignant pleural mesothelioma (MPM) is linked with poor prognosis, there is a lack of studies that functionally characterize stromal cells and tumor-infiltrating lymphocytes (TILs). Here, we aim to characterize the stromal subsets within MPM, investigate their relationship to TILs, and explore the potential therapeutic targets. Methods: We curated a core set of genes defining stromal/immune signatures expressed by mesenchymal cells within the TME using molecular analysis of The Cancer Genome Atlas (TCGA) MPM cohort. Stromal and immune profiles were molecularly characterized using flow cytometry, immunohistochemistry, microarray, and functionally evaluated using T cell-activation/expansion, coculture assays and drug compounds treatment, based on samples from an independent MPM cohort. Results: We found that a high extracellular matrix (ECM)/stromal gene signature, a high ECM score, or the ratio of ECM to an immune activation gene signature are significantly associated with poor survival in the MPM cohort in TCGA. Analysis of an independent MPM cohort (n = 12) revealed that CD8+ and CD4+ TILs were characterized by PD1 overexpression and concomitant downregulation in degranulation and CD127. This coincided with an increase in CD90+ cells that overexpressed PD-L1 and were enriched for ECM/stromal genes, activated PI3K-mTOR signaling and suppressed T cells. Protein array data demonstrated that MPM samples with high PD-L1 expression were most associated with activation of the mTOR pathway. Further, to reactivate functionally indolent TILs, we reprogrammed ex vivo TILs with Ibrutinib plus Rapamycin to block interleukin-2-inducible kinase (ITK) and mTOR pathways, respectively. The combination treatment shifted effector memory (T EM ) CD8+ and CD4+ TILs towards T cells that re-expressed CD45RA (T EMRA ) while concomitantly downregulating exhaustion markers. Gene expression analysis confirmed that Ibrutinib plus Rapamycin downregulated coinhibitory and T cell signature pathways while upregulating pathways involved in DNA damage and repair and immune cell adhesion and migration. Conclusions: Our results suggest that targeting the TME may represent a novel strategy to redirect the fate of endogenous TILs with the goal of restoring anti-tumor immunity and control of tumor growth in MPM., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Metastasis is a major contributor of death in cancer patients, and there is an urgent need for effective treatments of metastatic malignancies. Herein, ketoprofen (KP) and loxoprofen (LP) platinum(IV) complexes with antiproliferative and antimetastatic properties were designed and prepared by integrating chemotherapy and immunotherapy targeting cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and programmed death ligand 1 (PD-L1), besides DNA. A mono-KP platinum(IV) complex with a cisplatin core is screened out as a candidate possessing potent anti-proliferative and anti-metastasis activities both in vitro and in vivo. It induces serious DNA damage and further leads to high expression of γ-H2AX and p53. Moreover, it promotes apoptosis of tumor cells through mitochondrial apoptotic pathway Bcl-2/Bax/caspase3. Then, COX-2, MMP-9, NLRP3, and caspase1 as pivotal enzymes igniting inflammation and metastasis are obviously inhibited. Notably, it significantly improves immune response through restraining the expression of PD-L1 to increase CD3 + and CD8 + T infiltrating cells in tumor tissues.

High ERβ/HER oncogenic signaling defines lung tumors with an aggressive biology. We previously showed that combining the anti-estrogen fulvestrant with the pan-HER inhibitor dacomitinib reduced ER/HER crosstalk and produced synergistic anti-tumor effects in immunocompromised lung cancer models, including KRAS mutant adenocarcinoma. How this combination affects the tumor microenvironment (TME) is not known. We evaluated the effects of fulvestrant and dacomitinib on murine bone marrow-derived macrophages (BMDMs) and CD8+ T cells, and tested the efficacy of the combination in vivo, using the KRAS mutant syngeneic lung adenocarcinoma model, FVBW-17. While this combination synergistically inhibited proliferation of FVBW-17 cells, it had unwanted effects on immune cells, by reducing CD8+ T cell activity and phagocytosis in BMDMs and inducing PD-1. The effects were largely attributed to dacomitinib, which caused downregulation of Src family kinases and Syk in immune cells. In a subcutaneous flank model, the combination induced an inflamed TME with increased myeloid cells and CD8+ T cells and enhanced PD-1 expression in the splenic compartment. Concomitant administration of anti-PD-1 antibody with fulvestrant and dacomitinib was more efficacious than fulvestrant plus dacomitinib alone. Administering anti-PD-1 sequentially after fulvestrant plus dacomitinib was synergistic, with a two-fold greater tumor inhibitory effect compared to concomitant therapy, in both the flank model and in a lung metastasis model. Sequential triple therapy has potential for treating lung cancer that shows limited response to current therapies, such as KRAS mutant lung adenocarcinoma.