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Cell competition, a fitness-sensing process, is essential for tissue homeostasis. Using cancer metastatic latency models, we show that cell competition results in the displacement of latent metastatic (Lat-M) cells from the primary tumor. Lat-M cells resist anoikis and survive as residual metastatic disease. A memodeled extracellular matrix facilitates Lat-M cell displacement and survival in circulation. Disrupting cell competition dynamics by depleting secreted protein and rich in cysteine (SPARC) reduced displacement from orthotopic tumors and attenuated metastases. In contrast, depletion of SPARC after extravasation in lung-resident Lat-M cells increased metastatic outgrowth. Furthermore, multiregional transcriptomic analyses of matched primary tumors and metachronous metastases from patients with kidney cancer identified tumor subclones with Lat-M traits. Kidney cancer enriched for these Lat-M traits had a rapid onset of metachronous metastases and significantly reduced disease-free survival. Thus, an unexpected consequence of cell competition is the displacement of cells with Lat-M potential, thereby shaping metastatic latency and relapse. Significance: We demonstrate that cell competition within the primary tumor results in the displacement of Lat-M cells. We further show the impact of altering cell competition dynamics on metastatic incidence that may guide strategies to limit metastatic recurrences. This article is highlighted in the In This Issue feature, p. 1

Video shows displacement of Lat-M cells in cell competition. Lat-M cells (Green) surrounded by PA (Red) cells being displaced under cell competition assay conditions.

Cell competition, a fitness-sensing process, is essential for tissue homeostasis. Using cancer metastatic latency models, we show that cell competition results in the displacement of latent metastatic (Lat-M) cells from the primary tumor. Lat-M cells resist anoikis and survive as residual metastatic disease. A memodeled extracellular matrix facilitates Lat-M cell displacement and survival in circulation. Disrupting cell competition dynamics by depleting secreted protein and rich in cysteine (SPARC) reduced displacement from orthotopic tumors and attenuated metastases. In contrast, depletion of SPARC after extravasation in lung-resident Lat-M cells increased metastatic outgrowth. Furthermore, multiregional transcriptomic analyses of matched primary tumors and metachronous metastases from patients with kidney cancer identified tumor subclones with Lat-M traits. Kidney cancer enriched for these Lat-M traits had a rapid onset of metachronous metastases and significantly reduced disease-free survival. Thus, an unexpected consequence of cell competition is the displacement of cells with Lat-M potential, thereby shaping metastatic latency and relapse.Significance:We demonstrate that cell competition within the primary tumor results in the displacement of Lat-M cells. We further show the impact of altering cell competition dynamics on metastatic incidence that may guide strategies to limit metastatic recurrences.This article is highlighted in the In This Issue feature, p. 1

This document includes the followings: Supplementary Fig. S1-6 and legends Supplementary Table. S1-2 and legends Supplementary Video. S1 legend

Supplementary figures show the efficacy of TP-0903 and its effects on tumor infiltrating immune cells and EMT markers expression.

Pancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the United States, has a high metastatic rate, and is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, is a driver of metastasis and immune suppression in multiple cancer types. Here we use single-cell RNA-sequencing to reveal that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and that AXL expression correlates with classic markers of epithelial-to-mesenchymal transition. We demonstrate that AXL deficiency extends survival, reduces primary and metastatic burden, and enhances sensitivity to gemcitabine in an autochthonous model of PDA. PDA in AXL-deficient mice displayed a more differentiated histology, higher nucleoside transporter expression, and a more active immune microenvironment compared with PDA in wild-type mice. Finally, we demonstrate that AXL-positive poorly differentiated tumor cells are critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target in PDA.Implications:These studies implicate AXL as a marker of undifferentiated PDA cells and a target for therapy.

Supplementary Figure 1: 7rh reduces collagen-mediated signaling in a concentration-dependent manner in vivo; Supplementary Figure 2: 7rh inhibits Ddr1 activation in Pan02 tumors; Supplementary Figure 3: Inhibition of Ddr1 with 7rh does not induce observable toxicity; Supplementary Figure 4: 7rh in combination with chemotherapy reduced collagen deposition and AsPC-1 tumor weight; Supplementary Figure 5: 7rh in combination with chemotherapy reduced collagen deposition and KPC tumor weight

Supplementary Figure 1. (a) Representative images of metastasis to the liver of control mice in Colo357 model was shown by H&E staining. Total magnifications are 100X and 200X. Scale bar, 50μm. (b) Colo357 human pancreatic cancer cells (1 × 106) were injected orthotopically into the pancreas of SCID mice. Treatment began when the established tumor was visible by ultrasound, and consisted of control (saline, n = 6), gemcitabine 25 mg/kg twice weekly plus erlotinib 100 μg daily (standard of care, n = 6) as described in Kirane et al. (29), or mcr84 plus apricoxib (n = 10) and continued for 3 weeks. Tumor weight was normalized to the control and data from the treatment groups were compared. Data are displayed as mean {plus minus} SEM. *P < 0.05 vs. control, by ANOVA with Dunn's MCT. Supplementary Figure 2. Human pancreatic cancer cell lines, Colo357 (a) and AsPc-1 (b) were plated either under normal conditions or under conditions of forced EMT (treated with 20 ng/mL TGFβ on collagen I -coated plates for 72 hours). PGE2 levels were measured by ELISA after 500 nM apricoxib treatment. (c) Colo357 and AsPC-1 cells were plated under normal conditions or conditions of forced EMT (50 ng/ml TGFβ on collagen I -coated plates for 24 hours). Lysates were probed for the indicated targets by Western blotting. The induced EMT conditions promoted loss of E-Cadherin, gain of Vimentin expression. In Colo357, the expression of N-Cadherin was also upregulated by collagen I and TGFβ stimulation. β-actin was used as a loading control. Supplementary Figure 3. Microvessel density is not decreased by COX-2 inhibition. Supplementary Figure 4. KIC pancreatic tissues from the treated mice were subjected to immunohistochemistry Supplementary Figure 5. Anti-VEGF therapy results in reduced microvessel density, hypoxia induced TGFβ expression, epithelial plasticity and enhanced collagen deposition Table S1: List of antibodies

S1. Gene Ontology (GO) analysis of gene clusters from AXL-positive cancer cell populations based on scRNA-Seq data from Hosein et al (35). S2. Expression of Axl in tumors from WT and AXLLacZ/LacZ (AAKIC) KIC mice. S3. Tumor tissues from KIC and AxlLacZ/LacZ KIC mice were evaluated for indicated targets. S4. Flow cytometry of tumor-associated myeloid and lymphoid cells from KIC (n=11) and AxlLacZ/LacZ KIC (n=9). CD11b+ myeloid cells, CD4/CD3, and CD8/CD3 ratio were analyzed. 5. A) Cell proliferation was investigated in three different WT and AXL KO KPfC cells using MTS assay with eight technical replicates. B) Cell migration was investigated in three different WT and AXL KO KPfC cells by a "scratch" assay with three technical replicates. Monolayers of the indicated cells were wounded with a pipet tip. The cells were incubated in 1% serum media. Wound closure was monitored at 12, 24, and 36 hours and is reported as % wound closure. ***P < 0.001; ****P < 0.0001; by t-test. C) Colony formation (colonies/hpf) for three different WT and AXL KO KPfC cells with three technical replicates is shown by box plot (min to max). **P < 0.01; by t-test. S6. Expression of E-cadherin in WT and AXL KO KPfC cells. S7. A) WT and AxlLacZ/LacZ C57BL/6 mice orthotopically injected with 500,000 cells/mouse WT and AXL KO Pan02 cells were enrolled in a survival study. Groups were as follows: WT Pan02 cells in WT C57BL/6 mice (n=9); WT KPfC cells in AxlLacZ/LacZ C57BL/6 mice (n=7); AXL KO KPfC cells in WT C57BL/6 mice (n=6). B) 500,000 cells/mouse WT and AXL KO Pan02 cells were splenically injected into WT and AxlLacZ/LacZ C57BL/6 mice. Liver metastases were investigated 7 weeks after injection. Groups were as follows: WT Pan02 cells in WT C57BL/6 mice (n=8); WT KPfC cells in AxlLacZ/LacZ C57BL/6 mice (n=7); AXL KO KPfC cells in WT C57BL/6 mice (n=10); AXL KO KPfC cells in AxlLacZ/LacZ C57BL/6 mice (n=9). H&E and Alcian Blue stain were conducted in the liver tissues to confirm metastasis. Magnification, 10X. C) 1,000,000 cells/mouse WT and AXL KO Panc1 cells were orthotopically injected into NON SCID mice. Tumor weight were investigated 11 weeks after injection.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States with a 5-year survival less than 5%. Resistance to standard therapy and limited response to immune checkpoint blockade due to the immunosuppressive and stroma-rich microenvironment remain major challenges in the treatment of pancreatic cancer. A key cellular program involved in therapy resistance is epithelial plasticity, which is also associated with invasion, metastasis, and evasion of immune surveillance. The receptor tyrosine kinase AXL is a key driver of tumor cell epithelial plasticity. High expression and activity of AXL is associated with poor prognosis, metastasis, and therapy resistance in multiple types of cancer including pancreatic. Here, we show that an AXL inhibitor (TP-0903), has antitumor and therapy sensitizing effects in preclinical models of pancreatic ductal adenocarcinoma (PDA). We demonstrate that TP-0903 as a single agent or in combination with gemcitabine and/or anti-programmed cell death protein 1 (PD1) antibody has anti-metastatic and anti-tumor effects in PDA tumor bearing mice, leading to increased survival. In addition, gene expression analysis of tumors demonstrated upregulation of pro-inflammatory and immune activation genes in tumors from TP-0903-treated animals compared with the vehicle, indicating pharmacologic inhibition of AXL activation leads to an immunostimulatory microenvironment. This effect was augmented when TP-0903 was combined with gemcitabine and anti-PD1 antibody. These results provide clear rationale for evaluating TP-0903 in the treatment of pancreatic cancer.

Up to 50% of pancreatic ductal adenocarcinomas (PDA) harbor mutations in canonical TGFß pathway genes such as SMAD4 and TGFBR2. SMAD4 mutations have been shown to drive resistance to chemotherapy, metastasis, and margin positivity following surgical resection. To characterize the unique tumor microenvironment of SMAD4 mutant PDA, we developed isogenic, orthotopic mouse models using CRISPR mediated knockout of Smad4 in a cell line derived from KPfC (KrasLSL-G12D;Trp53fl/fl;Pdx1Cre) mice, a genetically engineered mouse model of pancreatic cancer. To characterize the alteration of the secretome caused by the loss of Smad4, we performed proteomic analysis with conditioned media collected from Smad4 wildtype and mutant cancer cell clones. Interestingly, the loss of Smad4 resulted in the production of neuroendocrine related factors. In addition, gene ontology analysis with the secretome suggested that these factors were associated with neuronal processes. These data indicate that loss of Smad4 in PDA cancer cells may lead to a neuroendocrine signature change. In particular, we found one of the most up-regulated secreted factors in the mutant cells was SPP1, which has been shown to promote tumor progression by affecting cancer cell stemness and macrophage polarization. To validate the proteomic results, we established orthotopic models with Smad4 wildtype and mutant clones. We found that loss of Smad4 resulted in larger tumor growth. Moreover, immunohistochemistry showed a significant up-regulation of SPP1 in the cancer cells of Smad4 mutant tumors. To further profile the alteration of immune landscape, we stained for markers of myeloid cells and lymphocytes. We found that Smad4 mutant tumors had an immunosuppressive microenvironment characterized by increased M2 macrophage, neutrophil and MDSC infiltration. Altogether, our study suggests that loss of SMAD4 in PDA may induce a neuroendocrine signature with increased SPP1 secretion in cancer cells. The production of this unique cancer cell secretome may result in a microenvironment marked by immunosuppressive myeloid cells. Citation Format: Gilbert Z. Murimwa, Francesca Rossi, Henry K. Fleming, Zeynep Yazgan, Dina Alzhanova, Huocong Huang, Rolf A. Brekken. Loss of Smad4 induces SPP1 secretion and immunosuppressive myeloid cell formation in pancreatic ductal adenocarcinoma. [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 3641.

Rhabdomyosarcoma (RMS) is a common soft tissue sarcoma in children that resembles the developing skeletal muscle. Unlike normal muscle cells, RMS cells fail to differentiate despite expression of the myogenic determination protein MYOD. The TWIST2 transcription factor is frequently overexpressed in fusion-negative RMS (FN-RMS). TWIST2 blocks differentiation by inhibiting MYOD activity in myoblasts, but its role in FN-RMS pathogenesis is incompletely understood. Here, we show that knockdown of TWIST2 enables FN-RMS cells to exit the cell cycle and undergo terminal myogenesis. TWIST2 knockdown also substantially reduces tumor growth in a xenograft model of FN-RMS. Mechanistically, TWIST2 controls H3K27 acetylation at distal enhancers by interacting with the chromatin remodelers SMARCA4 and CHD3 to activate growth-related and repress myogenesis-related target genes, respectively. These findings provide novel insights into the role of TWIST2 in maintaining an undifferentiated and tumorigenic state of FN-RMS and highlight the potential of suppressing TWIST2-regulated pathways to treat FN-RMS. Citation Format: Akansha M. Shah, Lei Guo, Maria Gabriella Morales, Priscilla Jaichander, Kenian Chen, Huocong Huang, Karla Cano Hernandez, Lin Xu, Rhonda Bassel-Duby, Eric N. Olson, Ning Liu. TWIST2 mediated chromatin remodeling promotes fusion-negative rhabdomyosarcoma. [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 4750.

PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.

Pancreatic ductal adenocarcinoma (PDA) is a devastating disease with a poor survival rate. It is resistant to therapy in part due to its unique tumor microenvironment, characterized by a desmoplastic reaction resulting in a dense stroma that constitutes a large fraction of the tumor volume. A major contributor to the desmoplastic reaction are cancer-associated fibroblasts (CAFs). CAFs actively interact with cancer cells and promote tumor progression by different mechanisms, including extracellular matrix deposition, remodeling, and secretion of tumor promoting factors, making CAFs an attractive target for PDA. However, emerging evidences indicate significant tumor-suppressive functions of CAFs, highlighting the complexity of CAF biology. CAFs were once considered as a uniform cell type within the cancer stroma. Recently, the existence of CAF heterogeneity in PDA has become appreciated. Due to advances in single cell technology, distinct subtypes of CAFs have been identified in PDA. Here we review recent updates in CAF biology in PDA, which may help develop effective CAF-targeted therapies in the future.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States with a 5-year survival less than 5%. Resistance to standard therapy and limited response to immune checkpoint blockade due to the immunosuppressive and stroma-rich microenvironment remain major challenges in the treatment of pancreatic cancer. A key cellular program involved in therapy resistance is epithelial plasticity, which is also associated with invasion, metastasis, and evasion of immune surveillance. The receptor tyrosine kinase AXL is a key driver of tumor cell epithelial plasticity. High expression and activity of AXL is associated with poor prognosis, metastasis, and therapy resistance in multiple types of cancer including pancreatic. Here, we show that an AXL inhibitor (TP-0903), has antitumor and therapy sensitizing effects in preclinical models of pancreatic ductal adenocarcinoma (PDA). We demonstrate that TP-0903 as a single agent or in combination with gemcitabine and/or anti-programmed cell death protein 1 (PD1) antibody has anti-metastatic and anti-tumor effects in PDA tumor bearing mice, leading to increased survival. In addition, gene expression analysis of tumors demonstrated upregulation of pro-inflammatory and immune activation genes in tumors from TP-0903-treated animals compared with the vehicle, indicating pharmacologic inhibition of AXL activation leads to an immunostimulatory microenvironment. This effect was augmented when TP-0903 was combined with gemcitabine and anti-PD1 antibody. These results provide clear rationale for evaluating TP-0903 in the treatment of pancreatic cancer.

Recent studies have identified a unique cancer-associated fibroblast (CAF) population termed antigen-presenting CAFs (apCAFs), characterized by the expression of major histocompatibility complex class II molecules, suggesting a function in regulating tumor immunity. Here, by integrating multiple single-cell RNA-sequencing studies and performing robust lineage-tracing assays, we find that apCAFs are derived from mesothelial cells. During pancreatic cancer progression, mesothelial cells form apCAFs by downregulating mesothelial features and gaining fibroblastic features, a process induced by interleukin-1 and transforming growth factor β. apCAFs directly ligate and induce naive CD4+ T cells into regulatory T cells (Tregs) in an antigen-specific manner. Moreover, treatment with an antibody targeting the mesothelial cell marker mesothelin can effectively inhibit mesothelial cell to apCAF transition and Treg formation induced by apCAFs. Taken together, our study elucidates how mesothelial cells may contribute to immune evasion in pancreatic cancer and provides insight on strategies to enhance cancer immune therapy. Citation Format: Huocong Huang, Zhaoning Wang, Yuqing Zhang, Rachana N. Pradhan, Debolina Ganguly, Raghav Chandra, Gilbert Murimwa, Steven Wright, Xiaowu Gu, Ravikanth Maddipati, Sören Müller, Shannon J. Turley, Rolf A. Brekken. Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C078.

Histologic features of diffuse-type gastric cancer indicate that the tumor microenvironment (TME) may substantially impact tumor invasiveness. However, cellular components and molecular features associated with cancer invasiveness in the TME of diffuse-type gastric cancers are poorly understood.We performed single-cell RNA-sequencing (scRNA-seq) using tissue samples from superficial and deep invasive layers of cancerous and paired normal tissues freshly harvested from five patients with diffuse-type gastric cancer. The scRNA-seq results were validated by immunohistochemistry (IHC) and duplexSeven major cell types were identified. Fibroblasts, endothelial cells, and myeloid cells were categorized as being enriched in the deep layers. Cell type-specific clustering further revealed that the superficial-to-deep layer transition is associated with enrichment in inflammatory endothelial cells and fibroblasts with upregulatedThis study reveals the spatial reprogramming of the TME that may underlie invasive tumor potential in diffuse-type gastric cancer. This TME profiling across tumor layers suggests new targets, such as CCL2, that can modify the TME to inhibit tumor progression in diffuse-type gastric cancer.

This commentary highlights the article by Ruggeri et al that reports the importance of discoidin domain receptor 1 in tissue homeostasis in pancreatic injury and pancreatic ductal adenocarcinoma pathogenesis.

Angiogenesis, a hallmark of cancer, is induced by vascular endothelial growth factor–A (hereafter VEGF). As a result, anti-VEGF therapy is commonly used for cancer treatment. Recent studies have found that VEGF expression is also associated with immune suppression in patients with cancer. This connection has been investigated in preclinical and clinical studies by evaluating the therapeutic effect of combining antiangiogenic reagents with immune therapy. However, the mechanisms of how anti-VEGF strategies enhance immune therapy are not fully understood. We and others have shown selective elevation of VEGFR2 expression on tumor-associated myeloid cells in tumor-bearing animals. Here, we investigated the function of VEGFR2+ myeloid cells in regulating tumor immunity and found VEGF induced an immunosuppressive phenotype in VEGFR2+ myeloid cells, including directly upregulating the expression of programmed cell death 1 ligand 1. Moreover, we found that VEGF blockade inhibited the immunosuppressive phenotype of VEGFR2+ myeloid cells, increased T cell activation, and enhanced the efficacy of immune checkpoint blockade. This study highlights the function of VEGFR2 on myeloid cells and provides mechanistic insight on how VEGF inhibition potentiates immune checkpoint blockade.

Recent studies have identified a unique cancer-associated fibroblast (CAF) population termed antigen-presenting CAFs (apCAFs), characterized by the expression of major histocompatibility complex class II molecules, suggesting a function in regulating tumor immunity. Here, by integrating multiple single-cell RNA-sequencing studies and performing robust lineage-tracing assays, we find that apCAFs are derived from mesothelial cells. During pancreatic cancer progression, mesothelial cells form apCAFs by downregulating mesothelial features and gaining fibroblastic features, a process induced by interleukin-1 and transforming growth factor β. apCAFs directly ligate and induce naive CD4

SummaryMultiple recent single cell RNA sequencing (scRNA-seq) studies have identified a unique cancer-associated fibroblast (CAF) population in pancreatic ductal adenocarcinoma (PDA) called antigen-presenting CAFs (apCAFs). apCAFs are characterized by the expression of MHC II molecules, suggesting a function in regulating tumor immunity. Here we integrated multiple scRNA-seq studies and found that apCAFs are derived from mesothelial cells. Our data show that during PDA progression, mesothelial cells form apCAFs by down-regulating mesothelial features and gaining the fibroblastic features, a process induced by IL-1 and TGFβ. Moreover, apCAFs directly ligate and induce naïve CD4+ T cells into regulatory T cells (Tregs) in an antigen-specific manner. Our study elucidates an important but neglected cell type in the regulation of PDA immunity and may lead to targeted therapeutic strategies that can overcome immune evasion in PDA.

Pancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the United States, has a high metastatic rate, and is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, is a driver of metastasis and immune suppression in multiple cancer types. Here we use single-cell RNA-sequencing to reveal that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and that AXL expression correlates with classic markers of epithelial-to-mesenchymal transition. We demonstrate that AXL deficiency extends survival, reduces primary and metastatic burden, and enhances sensitivity to gemcitabine in an autochthonous model of PDA. PDA in AXL-deficient mice displayed a more differentiated histology, higher nucleoside transporter expression, and a more active immune microenvironment compared with PDA in wild-type mice. Finally, we demonstrate that AXL-positive poorly differentiated tumor cells are critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target in PDA. Implications: These studies implicate AXL as a marker of undifferentiated PDA cells and a target for therapy.

Pancreatic ductal adenocarcinoma (PDAC) tumors are characterized by a desmoplastic reaction and dense collagen that is known to promote cancer progression. A central mediator of pro-tumorigenic collagen signaling is the receptor tyrosine kinase discoid domain receptor 1 (DDR1). DDR1 is a critical driver of a mesenchymal and invasive cancer cell PDAC phenotype. Previous studies have demonstrated that genetic or pharmacologic inhibition of DDR1 prevents PDAC tumorigenesis and metastasis. Here, we investigated whether DDR1 signaling has cancer cell non-autonomous effects that promote PDAC progression and metastasis. We demonstrate that collagen-induced DDR1 activation in cancer cells is a major stimulus for CXCL5 production, resulting in the recruitment of tumor-associated neutrophils (TANs), the formation of neutrophil extracellular traps (NETs) and subsequent cancer cell invasion and metastasis. Moreover, we have identified that collagen-induced CXCL5 production was mediated by a DDR1-PKCθ-SYK-NFκB signaling cascade. Together, these results highlight the critical contribution of collagen I-DDR1 interaction in the formation of an immune microenvironment that promotes PDAC metastasis.SummaryDeng et al find that collagen signaling via DDR1 on human pancreatic cancer cells drives production and release of the cytokine, CXCL5, into systemic circulation. CXCL5 then triggers infiltration of neutrophils into the tumor where they promote cancer cell progression.

Pancreatic ductal adenocarcinoma (PDA), a leading cause of cancer-related death in the US, has a high metastatic rate and is associated with persistent immune suppression. AXL, a member of the TAM (TYRO3, AXL, MERTK) receptor tyrosine kinase family, has been identified as a driver of metastasis and immune suppression in multiple cancer types. Here we use single cell RNA sequencing to reveal that AXL is expressed highly in tumor cells that have a mesenchymal-like phenotype and that AXL expression correlates with classic markers of mesenchymal tumor cells. We demonstrate that AXL-deficiency extends survival, reduces primary and metastatic burden and enhances sensitivity to gemcitabine in an autochthonous model of PDA. PDA in AXL-deficient mice displayed a more differentiated histology, higher nucleoside transporter expression and a more active immune microenvironment compared to PDA in wild-type mice. Finally, we demonstrate that AXL-positive mesenchymal tumor cells are critical for PDA progression and metastasis, emphasizing the potential of AXL as a therapeutic target for PDA patients.

Pancreatic ductal adenocarcinoma (PDA) and chronic pancreatitis are characterized by a dense collagen-rich desmoplastic reaction. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by collagens that can regulate cell proliferation, migration, adhesion, and remodeling of the extracellular matrix. To address the role of DDR1 in PDA, Ddr1-null (Ddr(−/−)) mice were crossed with the Kras(G12D/+); Trp53(R172H/+); Ptf1a(Cre/+) (KPC) model of metastatic PDA. Ddr1(−/−); KPC mice progress to differentiated PDA but resist progression to poorly differentiated cancer compared with KPC control mice. Strikingly, severe pancreatic atrophy accompanied tumor progression in Ddr1(−/−); KPC mice. To further explore the effects of Ddr1 ablation, Ddr1(−/−) mice were crossed with the Kras(G12D/+); Ptf1a(Cre/+) neoplasia model and subjected to cerulein-induced experimental pancreatitis. Similar to KPC mice, tissue atrophy was a hallmark of both neoplasia and pancreatitis models in the absence of Ddr1. Compared with controls, Ddr1(−/−) models had increased acinar cell dropout and reduced proliferation with no difference in apoptotic cell death between control and Ddr1(−/−) animals. In most models, organ atrophy was accompanied by increased fibrillar collagen deposition, suggesting a compensatory response in the absence of this collagen receptor. Overall, these data suggest that DDR1 regulates tissue homeostasis in the neoplastic and injured pancreas.

Transforming growth factor-β (TGFβ) is an important driver of tumor growth via intrinsic and extrinsic mechanisms, and is therefore an attractive target for developing cancer therapeutics. Using preclinical models, we characterized the anti-tumor activity of a small molecule inhibitor of TGFβ receptor I (TGFβRI), galunisertib (LY2157299 monohydrate). Galunisertib demonstrated potent and selective inhibition of TGFβRI with corresponding inhibition of downstream signaling via inhibition of SMAD phosphorylation (pSMAD). Galunisertib also inhibited TGFβ-induced pSMAD in vivo, which enabled a pharmacokinetic/pharmacodynamic profile in Calu6 and EMT6-LM2 tumors. Galunisertib demonstrated anti-tumor activity including inhibition of tumor cell migration and mesenchymal phenotype, reversal of TGFβ-mediated immune-suppression, and tumor growth delay. A concentration-effect relationship was established with a dosing schedule to achieve the optimal level of target modulation. Finally, a rat model demonstrated a correlation between galunisertib-dependent inhibition of pSMAD in tumor tissues and in PBMCs, supporting the use of PBMCs for assessing pharmacodynamic effects. Galunisertib has been tested in several clinical studies with evidence of anti-tumor activity observed in subsets of patients. Here, we demonstrate that galunisertib inhibits a number of TGFβ-dependent functions leading to anti-tumor activity. The enhanced understanding of galunisertib provides rationale for further informed clinical development of TGFβ pathway inhibitors.

Background & AimsPancreatic ductal adenocarcinoma (PDA) is a major cause of cancer-related death with limited therapeutic options available. This highlights the need for improved understanding of the biology of PDA progression. The progression of PDA is a highly complex and dynamic process featuring changes in cancer cells and stromal cells; however, a comprehensive characterization of PDA cancer cell and stromal cell heterogeneity during disease progression is lacking. In this study, we aimed to profile cell populations and understand their phenotypic changes during PDA progression.MethodsWe employed single-cell RNA sequencing technology to agnostically profile cell heterogeneity during different stages of PDA progression in genetically engineered mouse models.ResultsOur data indicate that an epithelial-to-mesenchymal transition of cancer cells accompanies tumor progression. We also found distinct populations of macrophages with increasing inflammatory features during PDA progression. In addition, we noted the existence of three distinct molecular subtypes of fibroblasts in the normal mouse pancreas, which ultimately gave rise to two distinct populations of fibroblasts in advanced PDA, supporting recent reports on intratumoral fibroblast heterogeneity. Our data also suggest that cancer cells and fibroblasts are dynamically regulated by epigenetic mechanisms.ConclusionThis study systematically outlines the landscape of cellular heterogeneity during the progression of PDA. It strongly improves our understanding of the PDA biology and has the potential to aid in the development of therapeutic strategies against specific cell populations of the disease.

Cancer can be conceptualized as arising from somatic mutations resulting in a single renegade cell escaping from the constraints of multicellularity. Thus, the era of precision medicine has led to intense focus on the cancer cell to target these mutations that result in oncogenic signaling and sustain malignancy. However, in pancreatic ductal adenocarcinoma (PDAC) there are only four abundantly common driver mutations (KRAS, CDKN2A, TP53, and SMAD4), which are not currently actionable. Thus, precision therapy for PDAC must look beyond the cancer cell. In fact, PDAC is more than a collection of renegade cells, instead representing an extensive, supportive ecosystem, having developed over several years, and consisting of numerous interactions between the cancer cells, normal mesenchymal cells, immune cells, and the dense extracellular matrix. In this issue, Huang and colleagues demonstrate how elucidation of these complex relationships within the tumor microenvironment (TME) can be exploited for therapeutic intervention in PDAC. They identify in a subset of PDAC with mutations in TGFβ signaling, that a paracrine signaling axis can be abrogated to modulate the TME and improve outcomes.

The most abundant proteins in vertebrates are collagens; this superfamily of proteins consists of 28 members that share a triple helical motif but are structurally and functionally diverse. The majority of collagens are deposited in the extracellular matrix (ECM) and can be bound by different classes of transmembrane receptors, including integrins, receptor tyrosine kinases such as discoidin domain receptors 1 and 2 (DDR1 and DDR2), glycoprotein VI, and the leukocyte-associated immunoglobulin-like receptor-1. Tumors develop and progress in the context of an ECM that is rich in collagens. Furthermore, collagen signaling in tumors contributes to tumor progression, metastasis, and resistance to chemotherapy. DDRs in particular are implicated in regulating tumor cell proliferation, migration, adhesion, and response to growth factors, and in promoting chemoresistance. As such, DDR inhibitors have been developed by different groups in an effort to inhibit collagen-mediated chemoresistance. This chapter provides an overview of the contribution of collagen and collagen signaling to tissue integrity and tumor progression and describes recent strategies to block collagen signaling to enhance chemosensitivity.

Immune profiling of tissue through multiplex immunohistochemistry is important for the investigation of immune cell dynamics, and it can contribute to disease prognosis and evaluation of treatment response in cancer patients. However, protocols for mouse formalin-fixed, paraffin-embedded tissue have been less successful. Given that formalin fixation and paraffin embedding remains the most common preparation method for processing mouse tissue, this has limited the options to study the immune system and the impact of novel therapeutics in preclinical models. In an attempt to address this, we developed an improved immunohistochemistry protocol with a more effective Ag-retrieval buffer. We also validated 22 Abs specific for mouse immune cell markers to distinguish B cells, T cells, NK cells, macrophages, dendritic cells, and neutrophils. In addition, we designed and tested novel strategies to identify immune cells for which unique Abs are currently not available. Last, in the 4T1 model of breast cancer, we demonstrate the utility of our protocol and Ab panels in the quantitation and spatial distribution of immune cells.

Vascular endothelial growth factor-A (VEGF) is the dominant angiogenic factor that promotes tumor neovascularization, making it a critical target for cancer therapy. VEGF binds and activates VEGF receptor 1 (VEGFR1) and VEGFR2, with VEGFR2 being the principal receptor mediating VEGF-induced angiogenesis while VEGFR1 has a negative regulatory effect on this process. Previously, we found that selective inhibition of VEGF binding to VEGFR2 with a fully human monoclonal antibody (r84) or its mouse chimeric counterpart (mcr84) is sufficient for effective control of tumor growth in preclinical models of breast cancer. However, accumulating evidence indicates that the effect of blocking VEGF activity in solid tumors extends beyond inhibition of angiogenesis. Concordantly, we have shown that VEGF blockade regulates immune cell trafficking across the endothelium by increasing the expression of adhesion molecules while decreasing FasL expression on tumor vasculature. In addition, specifically blocking VEGF binding to VEGFR2 promotes an immune stimulatory microenvironment by enhancing activation of CD8+ cytotoxic T cells as evidenced by elevated secretion of Granzyme B and IFN-γ. In addition, VEGF blockade also reduces FoxP3+ regulatory T cell infiltration in multiple murine models of cancer. We and others have shown that immune cells express receptors for VEGF. Our data indicate that VEGFR2 expression is elevated on myeloid cells selectively in tumor-bearing animals. Consistent with VEGFR2 expression, treatment with mcr84 results in a reduction in macrophage/myeloid cell infiltration into murine breast tumors. In conclusion, our results enhance understanding of the function of VEGFR2 signaling on myeloid cells and suggest that selective inhibition of VEGF from binding VEGFR2 facilitates immune activation landscape in breast cancer models, which might enhance the efficacy of immune checkpoint blockade. This abstract is also being presented as PO045. Citation Format: Yuqing Zhang, Huocong Huang, Noah Sorrelle, Rolf Brekken. Selective inhibition of VEGF binding to VEGFR2 promotes an immune stimulatory microenvironment in murine models of breast cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR011.

Pancreatic ductal adenocarcinomas are highly malignant cancers characterized by extensive invasion into surrounding tissues, metastasis to distant organs, and a limited response to therapy. A main feature of pancreatic ductal adenocarcinomas is desmoplasia, which leads to extensive deposition of collagen I. We have demonstrated that collagen I can induce epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. A hallmark of EMT is an increase in the expression of the mesenchymal cadherin N-cadherin. Previously we showed up-regulation of N-cadherin promotes tumor cell invasion and that collagen I-induced EMT is mediated by two collagen receptors, α2β1-integrin and discoidin domain receptor 1 (DDR1). DDR1 is a receptor-tyrosine kinase widely expressed during embryonic development and in many adult tissues and is also highly expressed in many different cancers. In the signaling pathway initiated by collagen, we have shown proline-rich tyrosine kinase 2 (Pyk2) is downstream of DDR1. In this study we found isoform b of DDR1 is responsible for collagen I-induced up-regulation of N-cadherin and tyrosine 513 of DDR1b is necessary. Knocking down Shc1, which binds to tyrosine 513 of DDR1b via its PTB (phosphotyrosine binding) domain, eliminates the up-regulation of N-cadherin. The signaling does not require a functional SH2 domain or the tyrosine residues commonly phosphorylated in Shc1 but is mediated by the interaction between a short segment of the central domain of Shc1 and the proline-rich region of Pyk2. Taken together, these data illustrate DDR1b, but not DDR1a, mediates collagen I-induced N-cadherin up-regulation, and Shc1 is involved in this process by coupling to both DDR1 and Pyk2.

Pancreatic ductal adenocarcinoma (PDA) remains one of the deadliest forms of cancer, in part, because it is largely refractory to current therapies. The failure of most standard therapies in PDA, as well as promising immune therapies, may be largely ascribed to highly unique and protective stromal microenvironments that present significant biophysical barriers to effective drug delivery, that are immunosuppressive, and that can limit the distribution and function of anti-tumor immune cells. Here, we utilized stromal re-engineering to disrupt these barriers and move the stroma toward normalization using a potent antifibrotic agent, halofuginone. In an autochthonous genetically engineered mouse model of PDA, halofuginone disrupted physical barriers to effective drug distribution by decreasing fibroblast activation and reducing key extracellular matrix elements that drive stromal resistance. Concomitantly, halofuginone treatment altered the immune landscape in PDA, with greater immune infiltrate into regions of low hylauronan, which resulted in increased number and distribution of both classically activated inflammatory macrophages and cytotoxic T cells. In concert with a direct effect on carcinoma cells, this led to widespread intratumoral necrosis and reduced tumor volume. These data point to the multifunctional and critical role of the stroma in tumor protection and survival and demonstrate how compromising tumor integrity to move toward a more normal physiologic state through stroma-targeting therapy will likely be an instrumental component in treating PDA.

Immune checkpoint blockade has achieved significant therapeutic success for a subset of cancer patients; however, a large portion of cancer patients do not respond. Unresponsive tumors are characterized as being immunologically "cold," indicating that these tumors lack tumor antigen-specific primed cytotoxic T cells. Sitravatinib is a spectrum-selective tyrosine kinase inhibitor targeting TAM (TYRO3, AXL, MerTK) and split tyrosine-kinase domain-containing receptors (VEGFR and PDGFR families and KIT) plus RET and MET, targets that contribute to the immunosuppressive tumor microenvironment. We report that sitravatinib has potent antitumor activity by targeting the tumor microenvironment, resulting in innate and adaptive immune cell changes that augment immune checkpoint blockade. These results suggest that sitravatinib has the potential to combat resistance to immune checkpoint blockade and expand the number of cancer patients that are responsive to immune therapy.

Resistance to standard therapy remains a major challenge in the treatment of pancreatic ductal adenocarcinoma (PDA). Although anti-VEGF therapy delays PDA progression, therapy-induced hypoxia results in a less differentiated mesenchymal-like tumor cell phenotype, which reinforces the need for effective companion therapies. COX-2 inhibition has been shown to promote tumor cell differentiation and improve standard therapy response in PDA. Here, we evaluate the efficacy of COX-2 inhibition and VEGF blockade in preclinical models of PDA. In vivo, the combination therapy was more effective in limiting tumor growth and metastasis than single-agent therapy. Combination therapy also reversed anti-VEGF–induced epithelial–mesenchymal transition and collagen deposition and altered the immune landscape by increasing tumor-associated CD8+ T cells while reducing FoxP3+ T cells and FasL expression on the tumor endothelium. Implications: Together, these findings demonstrate that COX-2 inhibition enhances the efficacy of anti-VEGF therapy by reducing hypoxia-induced epithelial plasticity and promoting an immune landscape that might facilitate immune activation. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/2/348/F1.large.jpg.

Epithelial-mesenchymal transition (EMT) is a developmental biological process that is hijacked during tumor progression. Cadherin switching, which disrupts adherens junctions and alters cadherin-associated signaling pathways, is common during EMT. In many tumors, substantial extracellular matrix (ECM) is deposited. Collagen is the most abundant ECM constituent and it mediates specific signaling pathways by binding to integrins and discoidin domain receptors (DDRs). The interaction of the collagen receptors results in activation of signaling pathways that promote tumor progression including an induction of the cadherin switching. DDR inhibitors have demonstrated anticancer therapeutic efficacy preclinically by inhibiting the collagen signaling. Understanding how collagen signaling impacts cellular processes including EMT and cadherin switching is of great interest especially given the strong interest in stromal targeted therapies for desmoplastic cancers.

The extracellular matrix (ECM), a principal component of pancreatic ductal adenocarcinoma (PDA), is rich in fibrillar collagens that facilitate tumor cell survival and chemoresistance. Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that specifically binds fibrillar collagens and has been implicated in promoting cell proliferation, migration, adhesion, ECM remodeling, and response to growth factors. We found that collagen-induced activation of DDR1 stimulated protumorigenic signaling through protein tyrosine kinase 2 (PYK2) and pseudopodium-enriched atypical kinase 1 (PEAK1) in pancreatic cancer cells. Pharmacologic inhibition of DDR1 with an ATP-competitive orally available small-molecule kinase inhibitor (7rh) abrogated collagen-induced DDR1 signaling in pancreatic tumor cells and consequently reduced colony formation and migration. Furthermore, the inhibition of DDR1 with 7rh showed striking efficacy in combination with chemotherapy in orthotopic xenografts and autochthonous pancreatic tumors where it significantly reduced DDR1 activation and downstream signaling, reduced primary tumor burden, and improved chemoresponse. These data demonstrate that targeting collagen signaling in conjunction with conventional cytotoxic chemotherapy has the potential to improve outcome for pancreatic cancer patients. Mol Cancer Ther; 16(11); 2473–85. ©2017 AACR.

Pancreatic ductal adenocarcinoma (PDAC), a leading cause of cancer-related death in the US, has a high metastatic rate that is associated with persistent immune suppression. Axl, a member of the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinase family, has been identified as a critical factor that drives metastasis and immune suppression in many cancer types. We have shown that pharmacological inhibition of Axl significantly reduces tumor progression and metastasis and improves response to chemotherapy in multiple preclinical PDAC models. Here we demonstrate that Axl-deficient PDAC bearing mice have longer median survival, smaller tumors, fewer metastases and higher sensitivity to chemotherapy (gemcitabine) compared to Axl wild-type (WT) PDAC mice. Furthermore, Axl-deficient PDAC displays a more differentiated histology and has a more inflammatory and active immune microenvironment. Single cell RNA sequencing of PDAC genetically engineered mouse models reveals that Axl is expressed highly in tumor cells that have a mesenchymal-like phenotype and Axl expression correlates with classic markers of mesenchymal tumor cells at the RNA and protein level. To investigate the importance of Axl on tumor cells, CRISPR-Cas9 was used to knockout Axl in PDAC cells. Human and murine Axl-deficient PDAC cells displayed a more differentiated (epithelial) phenotype in vitro including reduced migratory capacity compared to Axl WT parental cells. Orthotopic injection of Axl-deficient murine PDAC cells into Axl WT mice resulted in longer median survival and fewer metastases compared to WT mice injected with WT PDAC cells. However, Axl-deficient mice injected with WT PDAC cell injection showed similar median survival and metastases as WT mice injected with WT PDAC cells. These in vivo results indicate that Axl expression on tumor cells is critical for PDAC progression and metastasis. In vitro experiments using WT and Axl-deficient human PDAC cells show that Axl is required for TGFβ-mediated epithelial to mesenchymal transition, probably through regulating the expression of Smad2 and Smad4. Interesting, Axl depletion has no effect on the mRNA level of Smad2 and Smad4, but does decrease the protein level, indicating that Axl might be involved in post-translational regulation (e.g., protein stability) of canonical TGFβ signaling intermediates. In summary, our study demonstrates that Axl expressed by pancreatic cancer cells is critical to tumor cell plasticity, tumor progression and metastasis. Citation Format: Wenting Du, Huocong Huang, Jason Toombs, Zhaoning Wang, Abdel Hosein, Natalie Burton, Rolf A. Brekken. Axl is critical for pancreatic cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1037.

Pancreatic cancer is characterized by dense stroma consisting of myofibroblasts, abundant extracellular matrix (ECM) and inflammatory/immune cells. Elevated levels of transforming growth factor β (TGFβ), a prominent driver of stromal accumulation, is a negative prognostic indicator for pancreatic cancer patients. However, inhibition of TGFβ has been challenging due to the context-dependent tumor suppressor function of TGFβ in pancreatic cancer. We reported previously that neutralizing the activity of murine TGFβ receptor 2 (Tgfβr2) using a monoclonal antibody (2G8), has potent antitumor activity in orthotopic human pancreatic tumor xenografts. The findings in xenografts highlight that TGFβ induction of stromal cell function is critical for its impact on tumor cell behavior and PDA progression and supports that inhibition of TGFβ-dependent effector functions on stromal cells has potential efficacy in PDA. Herein we demonstrate that inhibition of murine Tgfβr2 reduces IL-6 production from cancer-associated fibroblasts (CAFs), resulting in a reduction of Stat3 activation in tumor cells and reversion of the immunosuppressive landscape of murine models of PDA. Moreover, the reduction of stromal IL-6 results in activation of NK cells. In summary, we found TGFβ induced IL-6 secretion from CAFs in PDA contributes to tumorigenesis and immunosuppression and can be abrogated by inhibition of stromal Tgfβr2. Citation Format: Huocong Huang, Moriah Hagopian, Wenting Du, Yuqing Zhang, Kyla E. Driscoll, Rolf A. Brekken. Targeted inhibition of Tgfβr2 reduces IL-6 production from cancer-associated fibroblasts, suppresses Stat3 activation in pancreatic cancer cells and reverses immunosuppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B68.

Loss of E-cadherin has been long considered to be a major hallmark of epithelial-mesenchymal transition (EMT) and has been reported in various cancers. P120 catenin regulates E-cadherin stability on the cell surface and also plays a role in intracellular signaling by modulating nuclear transcription. We recently characterized the nature of interactions between p120 catenin and Mucin 1 (MUC1) in pancreatic cancer. Expression of different p120 catenin isoforms with and without MUC1 induced distinct morphologies, cell adhesion, and dynamic properties of motility along with different metastatic properties in vivo. Re-expression of p120 catenin isoform 3A in the context of MUC1 expression in a p120 catenin-deficient cell line stabilized expression of E-cadherin. However, orthotopic implantation of tumors using this stable cell line produced large metastatic lesions to the liver, which exceeded the volume of the primary tumor, suggesting down regulation of E-cadherin is not required for tumor metastasis. Here we extend those studies by showing that ectopic expression of E-cadherin does not block in vitro invasion of the pancreatic cancer cells, and instead accelerated the rate of tumor invasion. Furthermore, results from 23 cases of human pancreatic primary tumor specimens revealed that most tumors exhibiting metastatic activity retained epithelial morphology and E-cadherin gene expression. Our results indicate that loss of E-cadherin and EMT are not required for metastasis and that an epithelial morphology can be maintained during the process of tumor cell movement.

The Six1 homeoprotein belongs to the Six (sine oculis) transcription factor family, the members of which are known to act as master regulators of development. Six1 is essential for promoting myogenesis during mammalian somitogenesis. Previous studies have shown that Six1 participates in later steps of myogenic differentiation by enhancing early activation of myogenin via binding to the Mef3 site of the myogenin promoter. In the present study, however, we show that overexpression of Six1 via retroviral infection suppresses the expression of myogenin and myosin in C2C12 myoblasts, consequently retarding myogenic differentiation without affecting cell proliferation or expression of Mef2 and Mef3. These findings further demonstrate the functional role of Six1 in myogenesis.