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One of the major reasons for poor cancer outcomes is the existence of cancer stem cells (CSCs). CSCs are a small subpopulation of tumor cells that can self-renew, differentiate into the majority of tumor cells, and maintain tumorigenicity. As CSCs are resistant to traditional chemotherapy and radiation, they contribute to metastasis and relapse. Thus, new approaches are needed to target and eliminate CSCs. Here, we sought to target and reduce the frequency of CSCs in melanoma by therapeutic vaccination against CSC-associated transcription factors, such as Sox2 and Nanog, and aldehyde dehydrogenase (ALDH). Toward this goal, we have identified novel immunogenic peptide epitopes derived from CSC-associated Sox2 and Nanog and synthesized synthetic high-density lipoprotein (sHDL) nanodisc vaccine formulated with Sox2, Nanog, and ALDH antigen peptides together with CpG, a Toll-like receptor 9 agonist. Vaccination with nanodiscs containing six CSC antigen peptides elicited robust T cell responses against CSC-associated antigens and promoted intratumoral infiltration of CD8+ T cells, while reducing the frequency of CSCs and CD4+ regulatory T cells within melanoma tumors. Nanodisc vaccination effectively reduced tumor growth and significantly extended animal survival without toxicity toward normal stem cells. Overall, our therapeutic strategy against CSCs represents a cost-effective, safe, and versatile approach that may be applied to melanoma and other cancer types, as well as serve as a critical component in combined therapies to target and eliminate CSCs.

Sentinel node biopsy (SLNB) is not routinely recommended for patients undergoing prophylactic mastectomy (PM), yet omission remains a subject of debate among surgeons. A modern patient cohort was examined to determine occult malignancy (OM) incidence within PM specimens to reinforce current recommendations.All PM performed over a 5-year period were retrospectively identified, including women with unilateral breast cancer who underwent synchronous or delayed contralateral PM or women with elevated cancer risk who underwent bilateral PM.The study population included 772 patients (598 CPM, 174 BPM) with a total of 39 OM identified: 17 invasive cancers (14 CPM, 3 BPM) and 22 DCIS (19 CPM, 3 BPM). Of the 86 patients for whom SLNB was selectively performed, 1 micrometastasis was identified. In the CPM cohort, risk of OM increased with age, presence of LCIS of either breast, or presence of a non-BRCA high-penetrance gene mutation, while preoperative magnetic resonance imaging was associated with lower likelihood of OM.Given the low incidence of invasive OM in this updated series, routine SLNB is of low value for patients undergoing PM. For patients with indeterminate radiographic findings, discordant preoperative biopsies, LCIS, or non-BRCA high-penetrance gene mutations, selective SLNB implementation could be considered.

Figure S1 indicated that ITGB4-targeted cancer immunotherapies did not cause severe immunotherapy-related toxicity.

Table S1. Yeast strains; Table S2. Primers used for yeasts mutagenesis; Table S3: B-factor for GTP/GDP during MD simulations with WT RRAGC or with point Mutations; Table S4: Energy contribution of each residue (kcal/mol) to GTP and GDP binding; Figure S1A: MD simulations of RRAGC (A)WT and (B-J) individual point mutations in complex with GDP (shown as sticks); Figure S1B: MD simulations of RRAGC (A)WT and (B-J) individual point mutations in complex with GTP (shown as sticks); Supplementary Figure S2: HA-Raptor immunoprecipitation out of transiently transfected HEK293T cells.

Purpose: This study was performed to further our understanding of the biological and genetic basis of follicular lymphoma and to identify potential novel therapy targets.Experimental Design: We analyzed previously generated whole exome sequencing data of 23 follicular lymphoma cases and one transformed follicular lymphoma case and expanded findings to a combined total of 125 follicular lymphoma/3 transformed follicular lymphoma. We modeled the three-dimensional location of RRAGC-associated hotspot mutations. We performed functional studies on novel RRAGC mutants in stable retrovirally transduced HEK293T cells, stable lentivirally transduced lymphoma cell lines, and in Saccharomyces cerevisiae.Results: We report recurrent mutations, including multiple amino acid hotspots, in the small G-protein RRAGC, which is part of a protein complex that signals intracellular amino acid concentrations to MTOR, in 9.4% of follicular lymphoma cases. Mutations in RRAGC distinctly clustered on one protein surface area surrounding the GTP/GDP–binding sites. Mutated RRAGC proteins demonstrated increased binding to RPTOR (raptor) and substantially decreased interactions with the product of the tumor suppressor gene FLCN (folliculin). In stable retrovirally transfected 293T cells, cultured in the presence or absence of leucine, multiple RRAGC mutations demonstrated elevated MTOR activation as evidenced by increased RPS6KB/S6-kinase phosphorylation. Similar activation phenotypes were uncovered in yeast engineered to express mutations in the RRAGC homolog Gtr2 and in multiple lymphoma cell lines expressing HA-tagged RRAGC-mutant proteins.Conclusions: Our discovery of activating mutations in RRAGC in approximately 10% of follicular lymphoma provides the mechanistic rationale to study mutational MTOR activation and MTOR inhibition as a potential novel actionable therapeutic target in follicular lymphoma. Clin Cancer Res; 22(21); 5383–93. ©2016 AACR.

Table S1 demonstrated that ITGB4-targeted immunotherapies with or without anti-PD-L1 did not significantly affect hematopoiesis.

Integrin β4 (ITGB4) has been shown to play an important role in the regulation of cancer stem cells (CSC). Immune targeting of ITGB4 represents a novel approach to target this cell population, with potential clinical benefit. We developed two immunologic strategies to target ITGB4: ITGB4 protein–pulsed dendritic cells (ITGB4-DC) for vaccination and adoptive transfer of anti-CD3/anti-ITGB4 bispecific antibody (ITGB4 BiAb)–armed tumor-draining lymph node T cells. Two immunocompetent mouse models were utilized to assess the efficacy of these immunotherapies in targeting both CSCs and bulk tumor populations: 4T1 mammary tumors and SCC7 head and neck squamous carcinoma cell line. Immunologic targeting of ITGB4 utilizing either ITGB4-DC or ITGB4 BiAb-T cells significantly inhibited local tumor growth and metastases in both the 4T1 and SCC7 tumor models. Furthermore, the efficacy of both of these ITGB4-targeted immunotherapies was significantly enhanced by the addition of anti–PD-L1. Both ITGB4-targeted immunotherapies induced endogenous T-cell cytotoxicity directed at CSCs as well as non-CSCs, which expressed ITGB4, and immune plasma–mediated killing of CSCs. As a result, ITGB4-targeted immunotherapy reduced not only the number of ITGB4high CSCs in residual 4T1 and SCC7 tumors but also their tumor-initiating capacity in secondary mouse implants. In addition, treated mice demonstrated no apparent toxicity. The specificity of these treatments was demonstrated by the lack of effects observed using ITGB4 knockout 4T1 or ITGB4-negative CT26 colon carcinoma cells. Because ITGB4 is expressed by CSCs across a variety of tumor types, these results support immunologic targeting of ITGB4 as a promising therapeutic strategy.Significance: This study identifies a novel mechanism of resistance to anti–PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients.

Phenotypic analysis of Pmel-1 splenocytes, thymocytes, ESCs (R1), and Pmel-1 iPSCs cultured on OP9 followed by OP9-DL1.

Relationship between MDSCs and breast cancer stemness

Supplementary Table S1: Primers used in this study

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Myeloid-derived suppressor cells (MDSC) contribute to immune suppression in cancer, but the mechanisms through which they drive metastatic progression are not fully understood. In this study, we show how MDSC convey stem-like qualities to breast cancer cells that coordinately help enable immune suppression and escape. We found that MDSC promoted tumor formation by enhancing breast cancer cell stem-like properties as well as by suppressing T-cell activation. Mechanistic investigations indicated that these effects relied upon cross-talk between the STAT3 and NOTCH pathways in cancer cells, with MDSC inducing IL6-dependent phosphorylation of STAT3 and activating NOTCH through nitric oxide leading to prolonged STAT3 activation. In clinical specimens of breast cancer, the presence of MDSC correlated with the presence of cancer stem-like cells (CSC) and independently predicted poor survival outcomes. Collectively, our work revealed an immune-associated mechanism that extrinsically confers cancer cell stemness properties and affects patient outcome. We suggest that targeting STAT3-NOTCH cross-talk between MDSC and CSC could offer a unique locus to improve cancer treatment, by coordinately targeting a coupled mechanism that enables cancer stemness and immune escape. Cancer Res; 76(11); 3156–65. ©2016 AACR.

Supplementary Materials and Methods

Correlations between CD33, ALDH1A1 and IL6 transcripts in patients with breast cancer

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MDSC phenotype and suppressor activity

Supplementary tables: Supplementary Table 1. Patient characteristics (All 3 cohorts) Supplementary Table 2. shRNA Sequences and Real-time PCR primers Supplementary Table 3. Multivariate analysis of overall survival (All 3 cohorts) Supplementary Table 4. Multivariate analysis of overall survival (Cohort 1, HER2+ patients) Supplementary Table 5. Multivariate analysis of overall survival (Cohorts 2 and 3, random patients)

Supplementary Figure 1 MDSC phenotype and suppressor activity Supplementary Figure 2 MDSCs in breast cancer tissues Supplementary Figure 3 Relationship between MDSCs and breast cancer stemness Supplementary Figure 4 MDSCs produce NO and IL-6 and induce tumor STAT3 phosphorylation Supplementary Figure 5 Correlations between CD33, ALDH1A1 and IL6 transcripts in patients with breast cancer

Cancer immunotherapies may be limited by their failure to target cancer stem cells (CSCs). We previously described an approach to target these cells using a dendritic cell (DC) vaccine primed with lysates of CSCs identified by aldehyde dehydrogenase (ALDH). However, its clinical application is limited by the difficulty of obtaining adequate amounts of tumor from patient to make CSC lysate for vaccine preparation. To address this issue, we evaluated targeting ALDH

Integrins mediate cell-cell interactions and communication with the extracellular matrix (ECM). These transmembrane protein receptors allow binding between a cell and its surroundings, initiating a breadth of intracellular signaling resulting in proliferation, differentiation, survival, or migration. Such responses have made integrins an attractive target for cancer therapy. Self-renewing and highly tumorigenic cancer stem cells (CSCs) are most resistant to traditional radiation treatment and chemotherapy, and therefore may contribute directly to the metastasis and relapse of the disease. In both the 4T1 mouse metastatic mammary tumor model and SCC7 head and neck squamous cell carcinoma model, integrin β4 (ITGB4) was expressed on ALDHhigh 4T1 and SCC7 CSCs. Using two immunological approaches, we targeted ITGB4 through 1) ITGB4 protein-pulsed dendritic cell (ITGB4-DC) vaccination or 2) via anti-CD3/anit-ITGB4 bispecific antibody (ITGB4 BiAb)-armed T cell adoptive transfer. These two therapies reduced ITGB4-expressing CSCs and inhibited local tumor growth and lung metastasis through ITGB4 specific cellular and humoral immune responses. Additionally, the combination of anti-PD-L1 immunotherapy with our two ITGB4-targeted approaches significantly improved treatment efficacy. We also found increased concentrations of serum IFN-γ and IL-6 in the 4T1 and SCC7 models which may help define future directions of this ITGB4-targeted study. Together, these results emphasize ITGB4 as a practical CSC immunological target with possible therapeutic benefits across tumor types with high ITGB4 expression.

We have conducted clinical trials for cancer immunotherapy using vaccine-primed lymph node (VPLN) T calls based on our animal studies with tumor-draining lymph node (TDLN) T cells. More recently we have identified antitumor effector TDLN B cells which demonstrated significant tumor reactivity in vitro and therapeutic efficacy in vivo upon adoptive transfer which can be enhanced by anti-PD-L1 administration. It is unknown whether tumor cells, particularly cancer stem cells (CSCs) could suppress the antitumor effector B cells. We found that depletion of B cells with anti-CD20 mAb prior to anti-PD-L1 administration resulted in more aggressive tumor growth, and the anti-tumor efficacy of anti-PD-L1 was significantly reduced in both 4T1 and CT26 murine tumor-bearing hosts, suggesting the involvement of host B cells in the anti-tumor effect of anti-PD-L1. In addition, administration of anti-PD-L1 partially recovered the humoral immune response, confirming the PD-L1/PD-1 pathway involvement in B cell suppression. We detected elevated expression of PD-1 on activated B cells, and higher PD-L1 expression on ALDHhigh CSCs than on ALDHlow non-CSCs. Co-culturing ALDHhigh CSCs with purified B cells significantly reduced IgG secretion, and such CSC-mediated B cell suppression was rescued by adding anti-PD-L1 mAb in a dose-dependent manner to the CSC-B cell co-culture. These experiments thus indicate that CSCs suppress the IgG production by B cells via the PD-L1/PD-1 axis. Tumor cells were recently recognized to suppress T cell function via secretion of exosomes that express PD-L1. To investigate if exosomal PD-L1 contributes to B cell immunosuppression, we generated Rab27ako D5 cells to delete exosomes as well as PD-L1ko D5 cells by CRISPR/Cas9 gene editing. D5-derived exosomes suppressed B cell proliferation and IgG production in vitro, and administration of these exosomes promoted tumor growth and reduced animal survival, revealing the role of tumor-derived exosomes in B cell immunosuppression. Tumor-derived exosomes expressed PD-L1, but PD-L1 was absent in the exosomes isolated from the PD-L1ko tumor cells as evident by western blot and flow cytometry. Deletion of Rab27a or PD-L1 did not affect the proliferation of the tumor cells in vitro. However, Rab27ako or PD-L1ko resulted in significantly enhanced host anti-tumor immunity evident by reduced 4T1 tumor growth in vivo compared with the WT tumor. Importantly, anti-PD-L1 therapy significantly reduced Rab27ako or PD-L1ko 4T1 tumor growth and prolonged animal survival vs. the WT tumor control. We conclude that PD-L1-expresing CSCs and PD-L1-expresing exosomes suppress antitumor effector B cells via the PD-L1/PD-1 axis. To characterize the suppression of CSC-derived exosomes on host B cells as well as T cells, isolation of Rab27ako ALDHhigh CSCs warranties further investigation. Citation Format: Ying Xin, Jiayu Liu, You Qin, Kexing Lyu, Sifei Yu, Ming Lin, Max Wicha, Alfred E. Chang, Qiao Li. Cancer stem cells and exosomal PD-L1 suppress anti-tumor B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2256.

Cancer stem cells (CSCs) proliferate extensively and drive tumor metastasis and recurrence. CSCs have been identified in over 20 cancer types to date, but it remains unknown how to target and eliminate CSCs in vivo. Aldehyde dehydrogenase (ALDH) is a marker that has been used extensively for isolating CSCs. Here we present a novel approach to target and reduce the frequency of ALDH(high) CSCs by vaccination against ALDH. We have identified ALDH1-A1 and ALDH1-A3 epitopes from CSCs and developed synthetic high-density lipoprotein nanodiscs for vaccination against ALDH(high) CSCs. Nanodiscs increased antigen trafficking to lymph nodes and generated robust ALDH-specific T cell responses. Nanodisc vaccination against ALDH(high) CSCs combined with anti-PD-L1 therapy exerted potent antitumor efficacy and prolonged animal survival in multiple murine models. Overall, this is the first demonstration of a simple nanovaccine strategy against CSCs and may lead to new avenues for cancer immunotherapy against CSCs.

Integrin β4 (ITGB4) has been shown to play an important role in the regulation of cancer stem cells (CSC). Immune targeting of ITGB4 represents a novel approach to target this cell population, with potential clinical benefit. We developed two immunologic strategies to target ITGB4: ITGB4 protein–pulsed dendritic cells (ITGB4-DC) for vaccination and adoptive transfer of anti-CD3/anti-ITGB4 bispecific antibody (ITGB4 BiAb)–armed tumor-draining lymph node T cells. Two immunocompetent mouse models were utilized to assess the efficacy of these immunotherapies in targeting both CSCs and bulk tumor populations: 4T1 mammary tumors and SCC7 head and neck squamous carcinoma cell line. Immunologic targeting of ITGB4 utilizing either ITGB4-DC or ITGB4 BiAb-T cells significantly inhibited local tumor growth and metastases in both the 4T1 and SCC7 tumor models. Furthermore, the efficacy of both of these ITGB4-targeted immunotherapies was significantly enhanced by the addition of anti–PD-L1. Both ITGB4-targeted immunotherapies induced endogenous T-cell cytotoxicity directed at CSCs as well as non-CSCs, which expressed ITGB4, and immune plasma–mediated killing of CSCs. As a result, ITGB4-targeted immunotherapy reduced not only the number of ITGB4high CSCs in residual 4T1 and SCC7 tumors but also their tumor-initiating capacity in secondary mouse implants. In addition, treated mice demonstrated no apparent toxicity. The specificity of these treatments was demonstrated by the lack of effects observed using ITGB4 knockout 4T1 or ITGB4-negative CT26 colon carcinoma cells. Because ITGB4 is expressed by CSCs across a variety of tumor types, these results support immunologic targeting of ITGB4 as a promising therapeutic strategy. Significance: This study identifies a novel mechanism of resistance to anti–PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients.

Aim: We aim to demonstrate that a local nanoparticle-mediated hyperthermia can effectively eliminate tumor-associated Tregs and thereby boost checkpoint blockade-based immunotherapy. Materials & methods: Photothermal therapy (PTT), mediated with systemically administered stealthy iron-oxide nanoparticles, was applied to treat BALB/c mice bearing 4T1 murine breast tumors. Flow cytometry was applied to evaluate both Treg and CD8+ T-cell population. Tumor growth following combination therapy of both PTT and anti-CTLA-4 was further evaluated. Results: Our data reveal that tumor-associated Tregs can be preferentially depleted via iron-oxide nanoparticles-mediated PTT. When combining PTT with anti-CTLA-4 immunotherapy, we demonstrate a significant inhibition of syngeneic 4T1 tumor growth. Conclusion: This study offers a novel strategy to overcome Treg-mediated immunosuppression and thereby to boost cancer immunotherapy.

Sentinel lymph node biopsy (SLNB) is increasingly utilized after neoadjuvant chemotherapy (NAC) in responsive adenopathy, particularly with placement of a marking clip in the involved node(s). This may allow a subset of patients to avoid axillary lymph node dissection. SLNB is still discouraged in inflammatory breast cancer (IBC). The purpose of this study is to examine the axillary pathologic complete response (AXpCR) in IBC patients with clinical adenopathy. There may be an implication to approach a subset of IBC patients for SLNB after NAC. A single-institution institutional review board-approved database was reviewed. Inclusion criteria were clinicopathologic diagnosis of IBC and age ≥ 18 years. Stage IV disease was excluded. We collected data on demographics, tumor characteristics including histology and subtype, axillary status, and treatment effect details. Sixty-six patients fulfilled criteria. Mean follow-up was 4.1 years. The AXpCR was 6% for luminal A and luminal B [human epidermal growth factor receptor (HER)2 −] subtypes, and 24% for basal subtype. The AXpCR rate was 64% for HER2-enriched and luminal B (HER2 +) patients. Achievement of AXpCR among these HER2-positive patients was statistically significant (p = 0.0001). There was minimal difference in achieving AXpCR in HER2-overexpressing patients regardless of hormone receptor status (p = 1.000). Understanding the best patients to select for use of SLNB or targeted lymph node dissection after treatment is evolving. This unique series identified and described the axillary pathologic characteristics of IBC patients following NAC. Further research is needed to confirm that the approach, axillary node clip placement prior to treatment, is feasible and accurate in IBC.

The value of chimeric antigen receptor-modified donor lymphocyte infusion (CAR-DLI) is unclear in B-cell acute lymphoblastic leukemia (B-ALL), particularly in patients with relapsed diseases after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, 5 B-ALL patients who relapsed after allo-HSCT received CAR-DLI (CAR-DLI group), and the outcome was compared with 27 relapsed B-ALL patients who received DLI therapy (DLI group). The median complete remission duration of CAR-DLI group was significantly (P=0.020) longer when compared with DLI group: 9 months (range, 2-29) versus 3.2 months (range, 0-17.4). Furthermore, patients receiving CAR-DLI showed significant (P=0.049) survival advantage over DLI group, with median overall survival of 12 months (range, 3-29) and 3.7 months (range, 0-65), respectively. Of note, no patient developed acute graft versus host disease in the CAR-DLI group, while incidence of acute graft versus host disease grades I-II and grades III-IV were 2 (7%) and 4 (14.8%) in the DLI group, respectively. In addition, cytokine release syndrome in CAR-DLI group was manageable. Overall, our study demonstrated that CAR-DLI significantly improved the survival of B-ALL patients relapsed after allo-HSCT, thus indicating that CAR-DLI may represent an alternative and more effective therapy for B-ALL patients with relapsed diseases.

We have reported that cancer stem cell (CSC) vaccination induces CSC-specific T and B cell responses associated with therapeutic efficacy which can be enhanced by anti-PD-L1 administration. Tumor cells have been recognized to suppress T cell function via secretion of exosomes that express PD-L1. Whether a similar suppression by CSCs on B cells exists is unknown. In this study, 4T1 (breast cancer) and CT26 (colon cancer) tumor-bearing mice were administered with anti-CD20 mAb to deplete B cells prior to anti-PD-L1 mAb administration. Depletion of B cells as compared to the controls resulted in more aggressive tumor growth, and the anti-tumor efficacy of anti-PD-L1 was significantly reduced for both 4T1 and CT26, indicating the involvement of host B cells in the anti-tumor effect of anti-PD-L1. Administration of anti-PD-L1 partially recovered the humoral immune response, confirming the PD-L1/PD-1 pathway involvement in B cell suppression. We detected elevated expression of PD-1 on activated B cells, and higher PD-L1 expression on ALDHhigh CSCs than on ALDHlow non-CSCs. We co-cultured purified B cells with ALDHhigh CSCs with or without the addition of anti-PD-L1 mAb and found that the reduction of IgG secretion of B cells suppressed by CSCs could be rescued by anti-PD-L1 in a dose-dependent manner. These experiments indicate that CSCs suppress the IgG production by B cells via the PD-L1/PD-1 axis. In a different model, we isolated exosomes from D5 (melanoma) tumor cell culture media by differential ultracentrifugation and verified the isolated exosomes by transmission electron microscopy and nanoparticle tracking analysis as well as by exosome markers: CD63, HRS, ALIX, and TSG101. We generated Rab27ako D5 and F10 (melanoma) cells to delete exosomes by the CRISPR/Cas9 gene editing system. Rab27a knockout significantly decreased the exosome production from D5 and F10 tumor cells. We also generated PD-L1ko D5 and F10 cells. While PD-L1 was expressed in both wild type tumor cells and tumor-derived exosomes, it was absent in the PD-L1ko D5 and F10 cells and the exosomes isolated from the PD-L1ko cells as evident by western blot and flow cytometry. Furthermore, IFNγ failed to augment the PD-L1 expression in D5 and F10 cells treated with PD-L1-targeted CRISPR/Cas9, confirming the knockout of the PD-L1 gene in these tumor cells. Deletion of Rab27a or PD-L1 did not affect the proliferation of the tumor cells. Knockout of PD-L1 did not influence exosome secretion, nor did the knockout of Rab27a influence cell-surface PD-L1 expression. Based on these findings, the use of Rab27ako and PD-L1ko D5 and F10 CSCs will allow us to further characterize the potential suppression of exosomal PD-L1 on host B cells as well as T cells. Citation Format: You Qin, Kexing Lyu, Sifei Yu, Ming Lin, Max Wicha, Alfred E. Chang, Qiao Li. Potential suppression of B effector cells by exosomal PD-L1 on anti-cancer stem cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2046.

BackgroundDendritic cells (DCs) are a promising therapeutic target in cancer immunotherapy given their ability to prime antigen-specific T cells, and initiate antitumor immune response. A major obstacle for DC-based immunotherapy is the difficulty to obtain a sufficient number of functional DCs. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSCs); however, therapeutic strategies to engage iPSC-derived DCs (iPSC-DCs) into cancer immunotherapy remain to be elucidated. Accumulating evidence showing that induction of tumor-residing DCs enhances immunomodulatory effect of radiotherapy (RT) prompted us to investigate antitumor efficacy of combining intratumoral administration of iPSC-DCs with local RT.MethodsMouse iPSCs were differentiated to iPSC-DCs on OP9 stromal cells expressing the notch ligand delta-like 1 in the presence of granulocyte macrophage colony-stimulating factor. Phenotype and the capacities of iPSC-DCs to traffic tumor-draining lymph nodes (TdLNs) and prime antigen-specific T cells were evaluated by flow cytometry and imaging flow cytometry. Antitumor efficacy of intratumoral injection of iPSC-DCs and RT was tested in syngeneic orthotopic mouse tumor models resistant to anti-PD-1 ligand 1 (PD-L1) therapy.ResultsMouse iPSC-DCs phenotypically resembled conventional type 2 DCs, and had a capacity to promote activation, proliferation and effector differentiation of antigen-specific CD8+ T cells in the presence of the cognate antigen in vitro. Combination of in situ administration of iPSC-DCs and RT facilitated the priming of tumor-specific CD8+ T cells, and synergistically delayed the growth of not only the treated tumor but also the distant non-irradiated tumors. Mechanistically, RT enhanced trafficking of intratumorally injected iPSC-DCs to the TdLN, upregulated CD40 expression, and increased the frequency of DC/CD8+ T cell aggregates. Phenotypic analysis of tumor-infiltrating CD8+ T cells and myeloid cells revealed an increase of stem-like Slamf6+ TIM3− CD8+ T cells and PD-L1 expression in tumor-associated macrophages and DCs. Consequently, combined therapy rendered poorly immunogenic tumors responsive to anti-PD-L1 therapy along with the development of tumor-specific immunological memory.ConclusionsOur findings illustrate the translational potential of iPSC-DCs, and identify the therapeutic efficacy of a combinatorial platform to engage them for overcoming resistance to anti-PD-L1 therapy in poorly immunogenic tumors.

Integrins mediate cell-cell interactions and communication with the extracellular matrix (ECM). These transmembrane protein receptors allow binding between a cell and its surroundings, initiating a breadth of intracellular signaling resulting in proliferation, differentiation, survival, or migration. Such responses have made integrins an attractive target for cancer therapy. Self-renewing and highly tumorigenic cancer stem cells (CSCs) are most resistant to traditional radiation treatment and chemotherapy, and therefore may contribute directly to the metastasis and relapse of the disease. In both the 4T1 mouse metastatic mammary tumor model and SCC7 head and neck squamous cell carcinoma model, integrin β4 (ITGB4) was expressed on ALDHhigh 4T1 and SCC7 CSCs. Using two immunological approaches, we targeted ITGB4 through 1) ITGB4 protein-pulsed dendritic cell (ITGB4-DC) vaccination or 2) via anti-CD3/anit-ITGB4 bispecific antibody (ITGB4 BiAb)-armed T cell adoptive transfer. These two therapies reduced ITGB4-expressing CSCs and inhibited local tumor growth and lung metastasis through ITGB4 specific cellular and humoral immune responses. Additionally, the combination of anti-PD-L1 immunotherapy with our two ITGB4-targeted approaches significantly improved treatment efficacy. We also found increased concentrations of serum IFN-γ and IL-6 in the 4T1 and SCC7 models which may help define future directions of this ITGB4-targeted study. Together, these results emphasize ITGB4 as a practical CSC immunological target with possible therapeutic benefits across tumor types with high ITGB4 expression.

BACKGROUND: The management of clinically node-positive breast cancer after neoadjuvant chemotherapy (NAC) has progressed with the potential to avoid the morbidity of axillary lymph node dissection in patients with complete response to therapy. This study addresses the impact of pretreatment nodal burden and tumor subtype on axillary pathologic complete response (AXpCR) in patients treated with NAC to better inform axillary surgical management. METHODS: A prospective database was reviewed to identify clinically node-positive patients who underwent NAC followed by axillary lymph node dissection. Patients were stratified in accordance with abnormal nodal burden on pretreatment axillary imaging defined as low (1–2 nodes) or high (3 nodes), and biologic subtype defined by hormone receptor (HR+, HR−) and HER2 (human epidermal growth factor receptor 2) status. The primary outcome was AXpCR. RESULTS: AXpCR was 43% in the study population. There was no difference in AXpCR between low and high nodal burden groups (44% versus 42%, P = 0.87). Subtype correlated to AXpCR (P < 0.001) with the highest rate (78%) in the HR−/HER2+ group. Overall, HER2+ patients had a significantly higher AXpCR than HER2− subtypes (66% versus 28% P < 0.001). HR and HER2 status were also predictive of AXpCR when comparing patient, tumor, and treatment variables. CONCLUSIONS: Biologic subtype better correlated with rates of AXpCR than nodal burden alone with the highest rates of AXpCR in HER2+ patients. Consideration of tumor biology is more informative than nodal burden when evaluating options for axillary management after NAC.

Summary of Key Points Gastrointestinal Perforation • Perforation affects approximately 20% of patients with acute abdominal emergencies. • Surgical intervention should be considered unless the patient's overall prognosis is poor. Gastrointestinal Bleeding • Bleeding affects approximately 15% of patients with acute abdominal emergencies. • Efficient correction of thrombocytopenia and coagulopathy may control the bleeding or expedite safe intervention. • Endoscopy or angiography can identify the source of bleeding and also provide therapeutic benefit. As such, nonoperative therapy is successful for many patients. Inflammatory Conditions • Neutropenic enterocolitis, often referred to as typhlitis, typically affects the terminal ileum, cecum, and ascending colon in patients with chemotherapy-induced neutropenia. Most patients respond to conservative management, but surgical intervention should be considered for those with perforation, uncontrolled sepsis, or persistent symptoms despite correction of neutropenia. • Anorectal conditions are seen in up to 8% of patients with cancer. Although the vast majority improves with medical therapy, patients with undrained infection or soft tissue necrosis often require surgical intervention. Gastrointestinal and Biliary Obstruction • Obstruction affects approximately 40% of patients with acute abdominal emergencies. • Functional status, overall prognosis, site(s) of obstruction, and goals of care are all important factors when deciding an optimal strategy to palliate a patient's malignant gastrointestinal (GI) obstruction. • Cross-sectional imaging provides critical information as to the etiology of malignant biliary obstruction and the technical feasibility of surgical intervention. Fistulae • GI and genitourinary fistulae are more often related to complications from surgery or radiation than they are to an underlying malignancy.

Immune checkpoint inhibitors and monoclonal antibodies reinvigorate cancer immunotherapy. However, these immunotherapies only benefit a subset of patients. We previously reported that ALDH tumor cells were highly enriched for cancer stem cells (CSCs), and ALDH CSC lysate-pulsed dendritic cell (CSC-DC) vaccine was shown to induce CSC-specific cytotoxic T lymphocytes. In this study, we investigated the CSC targeting effect of the CSC-DC vaccine combined with a dual blockade of programmed death-ligand 1 and cytotoxic T-lymphocyte-associated protein (CTLA-4) in B16-F10 murine melanoma tumor model. Our data showed that animals treated with the dual blockade of programmed death-ligand 1 and CTLA-4 and CSC-DC vaccine conferred significantly more tumor regression than the CSC-DC vaccine alone. Importantly, the triple combination treatment dramatically eliminated ALDH CSCs in vivo. We observed that CSC-DC vaccine in combination with anti-PD-L1 and anti-CTLA-4 administration resulted in ∼1.7-fold fewer PD-1CD8 T cells and ∼2.5-fold fewer CTLA-4CD8 T cells than the populations observed following the CSC-DC vaccination alone. Moreover, significant antitumor effects and dramatically eliminated ALDH CSCs following the triple combination treatment were accompanied by significantly enhanced T-cell expansion, suppressed transforming growth factor β secretion, enhanced IFN-γ secretion, and significantly enhanced host specific CD8 T-cell response against CSCs. Collectively, these data showed that administration of a-PD-L1 and a-CTLA-4 combined with CSC-DC vaccine may represent an effective immunotherapeutic strategy for cancer patients in clinical.

Using HER2-targeted antibody drug conjugates (ADC), we tested the therapeutic efficacy, direct impact on cancer stem cells (CSCs), and potential added benefit when combined with PD-L1 blockade in immunocompetent mouse tumor models. Two murine mammary tumor models were used in this study: D2F2/E2, a cell line resulting from stable transfection of parental D2F2 murine mammary tumor cells with wild-type human HER2, and 4T1-HER2, derived from parental 4T1 by stable transduction with wild-type human HER2. We demonstrate that HER2-targeted ADC treatment induced both cellular and humoral anti-HER2 adaptive immune responses which effectively targeted both bulk tumor and cancer stem cells. We recently investigated induction of host immunity using a tumor re-challenge strategy in tumor free animals subjected to ADC or ADC + anti-PD-L1 therapy. D2F2/E2 tumor-bearing mice were treated with ADC or ADC + anti-PD-L1 to generate tumor free mice. These tumor free animals were re-challenged with the relevant tumor D2F2/E2 or irrelevant tumor WT 4T1 respectively. While all the tumor free animals re-challenged with WT 4T1 showed aggressive tumor growth, ADC-treated tumor free animals re-challenged with D2F2/E2 demonstrated significantly (p Citation Format: You Qin, Hannah E. Dobson, Frank I. Comer, Alfred E. Chang, Max S. Wicha, Qiao Li. Specific anti-HER2 host immunity conferred by HER2-targeted antibody drug conjugate therapy and checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 514.

Although blockade of the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) immune checkpoint has revolutionized cancer treatment, how it works on tumor-infiltrating CD8(+) T cells recognizing the same antigen at various differentiation stages remains elusive. Here, we found that the chemokine receptor CX3CR1 identified 3 distinct differentiation states of intratumor CD8(+) T cell subsets. Adoptively transferred antigen-specific CX3CR1(–)CD8(+) T cells generated phenotypically and functionally distinct CX3CR1(int) and CX3CR1(hi) subsets in the periphery. Notably, expression of coinhibitory receptors and T cell factor 1 (Tcf1) inversely correlated with the degree of T cell differentiation defined by CX3CR1. Despite lower expression of coinhibitory receptors and potent cytolytic activity, in vivo depletion of the CX3CR1(hi) subset did not alter the antitumor efficacy of adoptively transferred CD8(+) T cells. Furthermore, differentiated CX3CR1(int) and CX3CR1(hi) subsets were impaired in their ability to undergo proliferation upon restimulation and had no impact on established tumors upon second adoptive transfer compared with the CX3CR1(–) subset that remained effective. Accordingly, anti–PD-L1 therapy preferentially rescued proliferation and cytokine production of the CX3CR1(–) subset and enhanced antitumor efficacy of adoptively transferred CD8(+) T cells. These findings provide a better understanding of the phenotypic and functional heterogeneity of tumor-infiltrating CD8(+) T cells and can be exploited to develop more effective immunotherapy.

Cancer stem cells (CSCs) are crucial for tumor recurrence and distant metastasis. Immunologically targeting CSCs represents a promising strategy to improve efficacy of multimodal cancer therapy. Modulating the innate immune response involving Toll-like receptors, macrophages, natural killer cells, and γδT cells has therapeutic effects on CSCs. Antigens expressed by CSCs provide specific targets for immunotherapy. CSC-primed dendritic cell-based vaccines have induced significant antitumor immunity as an adjuvant therapy in experimental models of established tumors. Targeting the tumor microenvironment CSC niche with cytokines or checkpoint blockade provides additional strategies to eliminate CSCs.

We previously tested HER2-targeted antibody drug conjugates (ADC) in immunocompromised (SCID) mice, precluding evaluation of host immunity, impact on cancer stem cells (CSCs), and potential benefit when combined with PD-L1 blockade. In this study, we tested HER2-targeted ADC in two immunocompetent mouse tumor models. HER2-targeted ADC specifically inhibited the growth of HER2-expressing tumors, prolonged animal survival, and reduced HER2(+) and PD-L1(+) cells. ADC plus anti-PD-L1 antibody augmented therapeutic efficacy, modulated immune gene signatures, increased the number and function of CD3(+) and CD19(+) tumor-infiltrating lymphocytes (TILs), induced tumor antigen specific immunological memory, stimulated B cell activation, differentiation, and IgG1 production both systemically and in the tumor microenvironment. In addition, ADC therapy modulated T cell subsets and their activation in TILs. Furthermore, HER2-targeted ADC reduced the number and tumorigenicity of ALDH(hi) CSCs. This study demonstrates that HER2-targeted ADC effectively targets ALDH(hi) CSCs and this effect is augmented by co-administration of anti-PD-L1 antibody.

Summary Objectives To establish the concept that the antigenicity/immunogenicity of ALDH high human head and neck squamous cell carcinoma (HNSCC) cancer stem cells (CSC) is distinct from that of ALDH low non-CSCs. Methods We generated CSC-loaded dendritic cells (DCs) to sensitize autologous peripheral blood T, B lymphocytes to react with CSCs using human HNSCC samples in vitro . Results From peripheral blood collected from patients with HNSCC, we obtained PBMCs. DCs generated from the PBMC and pulsed with the lysate of ALDH high cells isolated from cultured HNSCC cells (CSC-DC) could sensitize autologous T, B lymphocytes in vitro, which was evident by cytokine production, CTL activity, and antibody secretion of these primed T, B cells in response to ALDH high CSCs. In contrast, DCs pulsed with lysate of ALDH low cells (ALDH low -DC) resulted in limited sensitization/priming of autologous T, B lymphocytes to produce IFNγ, GM-CSF; lyse CSCs, and secrete IgM and IgG in response to ALDH high CSCs. These results demonstrated significant differences in the antigenicity/immunogenicity between ALDH high CSCs vs . ALDH low cells isolated from the tumor specimen of patients with HNSCC, which indicates the existence of unique CSC antigens in the ALDH high population. Conclusion It is feasible to generate DCs from the PBMCs and isolate ALDH high CSCs from cultured tumor cells of the patients with HNSCC to prepare CSC-DC vaccines that can induce anti-HNSCC CSC cellular and humoral immunity, indicating its potential clinical application to treat patients with HNSCC.

Current approaches to adoptive T-cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with useful in vivo characteristics. Theoretically, this limitation could be overcome by using induced pluripotent stem cells (iPSC) that could provide an unlimited source of autologous T cells. However, the therapeutic efficacy of iPSC-derived regenerated T cells remains to be demonstrated. Here, we report the first successful reprogramming of T-cell receptor (TCR) transgenic CD8(+) T cells into pluripotency. As part of the work, we established a syngeneic mouse model for evaluating in vitro and in vivo antitumor reactivity of regenerated T cells from iPSCs bearing a rearranged TCR of known antigen specificity. Stably TCR retained T-cell-derived iPSCs differentiated into CD4(+)CD8(+) T cells that expressed CD3 and the desired TCR in vitro Stimulation of iPSC-derived CD4(+)CD8(+) T cells with the cognate antigen in the presence of IL7 and IL15 followed by expansion with IL2, IL7, and IL15 generated large numbers of less-differentiated CD8(+) T cells with antigen-specific potent cytokine production and cytolytic capacity. Furthermore, adoptively transferred iPSC-derived CD8(+) T cells escaped immune rejection, mediated effective regression of large tumors, improved survival, and established antigen-specific immunological memory. Our findings illustrate the translational potential of iPSCs to provide an unlimited number of phenotypically defined, functional, and expandable autologous antigen-specific T cells with the characteristics needed to enable in vivo effectiveness. Cancer Res; 76(12); 3473-83. ©2016 AACR.

Exosome is a kind of tiny membrane vesicles containing small molecules such as mRNA, miRNA, and proteins. It was showed that exosomes participate in cancer progression and metastasis by transferring bioactive molecules between cancer and various cells in the tumor microenvironment. We isolated exosomes from murine melanoma D5 tumor cell culture media by differential ultracentrifuge, and verified the isolated exosomes by transmission electron microscopy and nanoparticle tracking analysis as well as by exosome markers, e.g. CD63, HRS, ALIX, and TSG101. Using these isolated exosomes, we tested their ability to promote tumor growth. C57BL/6J mice were inoculated with D5 cells on day 0, and were then divided into two groups: treated (i.v.) with PBS vs. PBS containing isolated exosomes every 3 days starting from day 7 for 2 weeks. We found that D5-derived exosomes considerably promoted the tumor growth and notably reduced animal survival. It was reported recently that exosomes could function as immunosuppressive factors in immune responses. To test this in our animal models, we generated Rab27ako D5 and F10 cells to delete exosomes by CRISPR/Cas9 gene editing system. Rab27a knockout significantly decreased the exosome production from D5 and F10 tumor cells as demonstrated by nanoparticle tracking analysis. In addition, it was also reported that exosomes mediate immune suppression via its expression of PD-L1. To address this issue in our animal models, we also generated PD-L1ko D5 and F10 cells. While PD-L1 was expressed in both wild type tumor cells and tumor-derived exosomes, it was absent in the PD-L1ko D5 and F10 cells and the exosomes isolated from the PD-L1ko cells as evident by western blot and flow cytometry. Furthermore, IFNγ failed to augment the PD-L1 expression in D5 and F10 cells treated with PD-L1-trageted CRISPR/Cas9, confirming the knockout of PD-L1 gene in these tumor cells. Deletion of Rab27a or PD-L1 did not affect the proliferation of the tumor cells. Knockout of PD-L1 did not influence exosome secretion, nor did the knockout of Rab27a influence cell-surface PD-L1 expression. Although the immune suppression of exosome on T cells is well-known, such suppression of exosome on other immune cells is largely unknown. Based on our initial experimental results as described above, utilizing the Rab27ako and PD-L1ko D5 and F10 cells generated in our lab will allow us to evaluate the potential suppression of exosomal PD-L1 on host T cells as well as other immune components involved in cancer immunotherapy. Citation Format: You Qin, Kexing Lyu, Sifei Yu, Ming Lin, Max Wicha, Alfred E. Chang, Qiao Li. Generation of Rab27ako and PD-L1ko tumor cells to evaluate potential suppression of exosomal PD-L1 on host immune components involved in cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2157.

In the past decades, chemotherapy has shown encouraging but limited efficacy treating triple negative breast cancer (TNBC). Targeting cancer stem cells (CSCs) has been recognized as a promising method to inhibit tumor growth and dissemination. This study aimed to determine whether an anti-psychotic drug thioridazine (THZ) could sensitize metastatic TNBC cells to anti-cancer drug carboplatin (CBP). To this end, metastatic TNBC (4T1)-bearing animals were treated with THZ, CBP and their combination respectively. MTT, flow cytometry, electron microscope and western blot were performed to evaluate the treated 4T1 cells. Tumor size and lung metastasis were monitored. The percentage of residual 4T1 cancer stem cells and the tumorigenecity of residual tumor cells from each treated group were compared. We found that both mono- and combination treatments suppressed 4T1 cells and induced apoptosis in a dose-dependent manner. These effects involved mitochondrial dysfunction and mitochondria-mediated apoptosis. The structure of mitochondrial membrane was impaired as observed by electron microscope, and the mitochondrial membrane stability was interrupted as evident by down-regulated Bcl-2 & Bcl-xL expression, and up-regulated BAX expression. Both extrinsic and intrinsic apoptosis were demonstrated by enrichment of activated caspase 3, 8, 9. Tumor growth and lung metastasis in 4T1-bearing animals were markedly inhibited by CBP combined with THZ. We also found suppression of PI3k/AKT/mTOR pathway and activation of ER stress by CSP + THZ both in vitro and in vivo. Moreover, angiogenesis was inhibited, demonstrating an important mechanism for tumor metastasis inhibition. More importantly, THZ demonstrated the capability of destroying CSCs and this effect was amplified when combined with CBP. The tumorigenecity of CBP + THZ combination-treated residual tumor cells was significantly impaired. These results show that THZ could inhibit 4T1 tumor cell proliferation and induce apoptosis and significantly destroy the stability of mitochondrial membrane, causing extrinsic and intrinsic apoptosis when combined with CBP. A signature of dysangiogenesis indicated that angiogenesis might be involved in this process. These findings suggest that the anti-psychotic drug THZ is a promising chemosensitizer in TNBC tumor and TNBC patients may benefit from THZ + CBP administration treatment by targeting cancer stem cells. Keywords: Triple-negative breast cancer (TNBC); cancer stem cells (CSCs); thioridazine (THZ); carboplatin (CBP). Citation Format: Yi Wang, Leiming Xia, Li Gong, Yang Xia, Liu Liu, Yuanyuan Liu, Wicha Max, Alfred E. Chang, Qiao Li, Yangyi Bao. Synergistic inhibition of thioridazine combined with carboplatin on triple negative breast cancer by targeting cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3803.

Dendritic cell (DC)-based vaccines designed to target cancer stem cells (CSC) can induce significant antitumor responses via conferring host anti-CSC immunity. Our recent studies have demonstrated that CSC-DC vaccine could inhibit metastasis of primary tumors and induce humoral immune responses against cancer stem cells. This approach highlights the promise of cancer stem cell vaccine in cancer immunotherapy.

While SLN biopsy is recommended for melanoma ≥1 mm in depth, its use among the elderly population is more controversial. We reviewed our experience at the University of Michigan with melanoma patients ≥75 years of age. A total of 952 melanoma patients ≥75 years of age from 1996 to 2011 were identified from our institutional review board-approved database. In addition to clinicopathologic features and outcome data, comorbidity data were collected to calculate the Charlson comorbidity index (CCI). Univariate and multivariate Cox regression analysis was performed to characterize predictors of outcome. Kaplan–Meier analysis was used to generate survival curves. Among 553 clinically node-negative patients with melanoma ≥1 mm in Breslow thickness, 213 had wide excision alone, whereas 340 had excision and SLN biopsy, with 83 (24 %) having a positive SLN. SLN biopsy was less likely with older age (p