Cancer-associated Fibroblasts Cells Research Articles

Combined MEK and STAT3 Inhibition Uncovers Stromal Plasticity by Enriching for Cancer-Associated Fibroblasts With Mesenchymal Stem Cell-Like Features to Overcome Immunotherapy Resistance in Pancreatic Cancer

We have shown that reciprocally activated rat sarcoma (RAS)/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and Janus kinase/signal transducer and activator of transcription 3 (STAT3) pathways mediate therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC), while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance and alters stromal architecture. We now determine whether MEKi+STAT3i reprograms the cancer-associated fibroblast (CAF) and immune microenvironment to overcome resistance to immune checkpoint inhibition in PDAC. CAF and immune cell transcriptomes in MEKi (trametinib)+STAT3i (ruxolitinib)-treated vs vehicle-treated Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) tumors were examined via single-cell RNA sequencing (scRNAseq). Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 silencing of CAF-restricted Map2k1/Mek1 or Stat3, or both, enabled interrogation of CAF-dependent effects on immunologic remodeling in orthotopic models. Tumor growth, survival, and immune profiling via mass cytometry by time-of-flight were examined in PKT mice treated with vehicle, anti-programmed cell death protein 1 (PD-1) monotherapy, and MEKi+STAT3i combined with anti-PD1. MEKi+STAT3i attenuates Il6/Cxcl1-expressing proinflammatory and Lrrc15-expressing myofibroblastic CAF phenotypes while enriching for Ly6a/Cd34-expressing CAFs exhibiting mesenchymal stem cell-like features via scRNAseq in PKT mice. This CAF plasticity is associated with M2-to-M1 reprogramming of tumor-associated macrophages, and enhanced trafficking of cluster of differentiation 8+ T cells, which exhibit distinct effector transcriptional programs. These MEKi+STAT3i-induced effects appear CAF-dependent, because CAF-restricted Mek1/Stat3 silencing mitigates inflammatory-CAF polarization and myeloid infiltration invivo. Addition of MEKi+STAT3i to PD-1 blockade not only dramatically improves antitumor responses and survival in PKT mice but also augments recruitment of activated/memory T cells while improving their degranulating and cytotoxic capacity compared with anti-PD-1 monotherapy. Importantly, treatment of a patient who has chemotherapy-refractory metastatic PDAC with MEKi (trametinib), STAT3i (ruxolitinib), and PD-1 inhibitor (nivolumab) yielded clinical benefit. Combined MEKi+STAT3i mitigates stromal inflammation and enriches for CAF phenotypes with mesenchymal stem cell-like properties to overcome immunotherapy resistance in PDAC.

Cancer-Associated Fibroblasts in a 3D Engineered Tissue Model Induce Tumor-like Matrix Stiffening and EMT Transition.

Simple SummaryThe physical properties of a tumor, such as stiffness, are important drivers of tumor progression. However, current in vitro tumor models fail to recapitulate the full range of physical properties observed in solid tumors. Here, we proposed a 3D self-assembly engineered bladder model using cancer-associated fibroblasts in which stromal cells produce their extracellular matrix. We then proceeded to assess how our model recapitulates biological and mechanical features found in tumors. We confirmed that stroma assembled by cancer-associated fibroblasts have increased extracellular matrix content and display increased remodeling and higher stiffness. Moreover, normal urothelial cells seeded on the tumor model displayed a mechanotransduction response, increased cell proliferation, cell infiltration within stroma, and displayed features of the epithelial-to-mesenchymal transition. Altogether, we demonstrated that our cancer-associated fibroblast-derived tumor stroma recapitulates several biological and physical features expected from a tumor-like environment and, thus, provides the basis for more accurate cancer models.A tumor microenvironment is characterized by its altered mechanical properties. However, most models remain unable to faithfully recreate the mechanical properties of a tumor. Engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture and composition. Here, we used the self-assembly method based on a bladder tissue model to engineer a tumor-like environment. The tissue-engineered tumor models were reconstituted from stroma-derived healthy primary fibroblasts (HFs) induced into cancer-associated fibroblast cells (iCAFs) along with an urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be stiffer, with increased ECM deposition and remodeling. The urothelial cells overlaid on the iCAFs-derived ECM were more contractile, as measured by quantitative polarization microscopy, and displayed increased YAP nuclear translocation. We further showed that the proliferation and expression of epithelial-to-mesenchymal transition (EMT) marker in the urothelial cells correlate with the increased stiffness of the iCAFs-derived ECM. Our data showed an increased expression of EMT markers within the urothelium on the iCAFs-derived ECM. Together, our results demonstrate that our tissue-engineered tumor model can achieve stiffness levels comparable to that of a bladder tumor, while triggering a tumor-like response from the urothelium.

Interplay Between Immune and Cancer-Associated Fibroblasts: A Path to Target Metalloproteinases in Penile Cancer.

Extracellular matrix (ECM) remodeling and inflammation have been reported in penile carcinomas (PeCa). However, the cell types and cellular crosstalk involved in PeCa are unexplored. We aimed to characterize the complexity of cells and pathways involved in the tumor microenvironment (TME) in PeCa and propose target molecules associated with the TME. We first investigated the prognostic impact of cell types with a secretory profile to identify drug targets that modulate TME-enriched cells. The secretome analysis using the PeCa transcriptome revealed the enrichment of inflammation and extracellular matrix pathways. Twenty-three secreted factors were upregulated, mainly collagens and matrix metalloproteinases (MMPs). The deregulation of collagens and MMPs was confirmed by Quantitative reverse transcription - polymerase chain reaction (RT-qPCR). Further, the deconvolution method (digital cytometry) of the bulk samples revealed a high proportion of macrophages and dendritic cells (DCs) and B cells. Increased DCs and B cells were associated with better survival. A high proportion of cancer-associated fibroblasts (CAFs) was observed in low-survival patients. Patients with increased CAFs had decreased immune cell proportions. The treatment with the MMP inhibitor GM6001 in CAF cells derived from PeCa resulted in altered cell viability. We reported a crosstalk between immune cells and CAFs, and the proportion of these cell populations was associated with prognosis. We demonstrate that a drug targeting MMPs modulates CAFs, expanding the therapeutic options of PeCa.

MIF confers survival advantage to pancreatic CAFs by suppressing interferon pathway-induced p53-dependent apoptosis.

The presence of activated pancreatic stellate cells (PSCs) in the pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a significant role in cancer progression. Macrophage migration inhibitory factor (MIF) is overexpressed in PDAC tissues and expressed by both cancer and stromal cells. The pathophysiological role of MIF in PDAC-associated fibroblasts or PSCs is yet to be elucidated. Here we report that the PSCs of mouse or cancer-associated fibroblast cells (CAFs) of human expresses MIF and its receptors, whose expression gets upregulated upon LPS or TNF-α stimulation. In vitro functional experiments showed that MIF significantly conferred a survival advantage to CAFs/PSCs upon growth factor deprivation. Genetic or pharmacological inhibition of MIF also corroborated these findings. Further, co-injection of mouse pancreatic cancer cells with PSCs isolated from Mif-/- or Mif+/+ mice confirmed the pro-survival effect of MIF in PSCs and also demonstrated the pro-tumorigenic role of MIF expressed by CAFs in vivo. Differential gene expression analysis and in vitro mechanistic studies indicated that MIF expressed by activated CAFs/PSCs confers a survival advantage to these cells by suppression of interferon pathway induced p53 dependent apoptosis.

CAFs-derived small extracellular vesicles circN4BP2L2 promotes proliferation and metastasis of colorectal cancer via miR-664b-3p/HMGB3 pathway

ABSTRACT Our previous research has demonstrated that colorectal cancer (CRC) progression was promoted by circN4BP2L2. This study aimed to further explore the mechanism of circN4BP2L2 in the development of CRC from the perspective of small extracellular vesicles (sEVs). Cancer-associated fibroblasts cell (CAFs) and normal fibroblasts cell (NFs) were isolated from CRC tissues and adjacent tissues, respectively. The ultra-centrifugation was used for extraction of their related sEVs. Cell proliferation and apoptosis were analyzed using CCK-8 and flow cytometry, respectively. Transwell assay was conducted to measure cell migration. The tube formation ability was assessed by tube formation assay. The target relationships between circN4BP2L2 and miR-664b-3p, and miR-664b-3p and HMGB3 were validated by dual-luciferase reporter detection. The effect of CAFs-derived sEV (CAFs-sEVs) circN4BP2L2 on CRC was further studied in nude mice. CAFs-exo promoted cell proliferation, migration, tube formation ability, and inhibited apoptosis of CRC cells. CAFs-sEV circN4BP2L2 knockdown reversed the above results. CircN4BP2L2 directly targeted miR-664b-3p, and HMGB3 was targeted by miR-664b-3p. Moreover, subcutaneous tumorigenesis and liver metastasis of nude mice with CRC were repressed by CAFs-sEV circN4BP2L2 knockdown. CAFs-sEV circN4BP2L2 knockdown restrained CRC cell proliferation and migration by regulating miR-664b-3p/HMGB3 pathway.

Abstract 3197: Blockade of regulatory signaling pathways reduces immunosuppression in the PDAC tumor microenvironment

Abstract Pancreatic cancer (PDAC) has the highest mortality rate of all major cancers, with few treatment options that provide long-term benefit to patients. Cancer associated fibroblasts (CAFs), the primary cellular component of the pancreatic tumor stroma, play a key role in the formation of an immunosuppressive tumor microenvironment (TME). A better understanding of the disease biology and cellular pathways that could be targeted for treatment of PDAC are needed. To investigate mechanisms driving immune suppression and pathways that might be targetable for treatment in PDAC, we used models where cross-talk between stromal and tumor cells could be evaluated. Three PDAC tumor cell lines were co-cultured (CC) with primary CAFs from multiple donors for 3 days, and secretion of numerous immunosuppressive cytokines, including IL6, IL8, IL10, CXCL12, was found to be upregulated in CC vs single culture (SC). IL6 and IL8 were shown to increase 2-38-fold in CC vs SC CAFs, and 1.5-20,000-fold vs SC PANCs, dependent upon cell line. By whole genome array, an upregulation of genes involved in NFkB, IL6, IL8, and AKT signaling was also observed (-log(p-value)>4) in CAF cells following CC. Multiple pathways have been implicated in regulating the immunosuppressive PDAC TME, including: CD73, a critical mediator of adenosine formation which is known to alter immune activity; and IL1, which is secreted by PDAC tumor cells, known to increase inflammation in the TME, and promote PDAC progression. The effects of CD73 inhibition and IL1R inhibition were evaluated to better characterize the downstream effects of these pathways on regulating tumor immunosuppression. Inhibition of CD73 in the CC reduced production of the immunosuppressive cytokines IL6, IL8, MCP1 CXCL12, IL1a, IL1b, IL10 (16-59%). Preliminary genomic studies indicated that inhibition of CD73 decreased genes involved in invasion, migration, metastasis, EMT and angiogenesis, as well as increasing genes involved in type 1 IFN activation, including OAS1, DDX60, IFIT1 and IRF7 (FC 1.5-2). Similarly, blockade of IL1R (which is highly expressed on PDAC CAFs and is the receptor for both IL1a and IL1b) reduced immune suppressive cytokine production from PDAC tumor conditioned media-stimulated CAFs, e.g. lowering secretion of IL6 and IL8 (4-20-fold) in a dose-dependent manner. Blocking IL1R in a PDAC dissociated tumor cell (DTC) model resulted in decreased secretion of CXCL1 and IL6 (60-75% over 3-6 days in culture), and an upregulation of IFNg (2-10-fold) in this culture by d3. These results suggest that a blockade of regulatory signaling pathways may be useful to reduce immune suppression and return the PDAC TME to a phenotype more amenable to immune checkpoint inhibition. Further characterization of these pathways may lead to new therapeutic combination approaches for pancreatic cancer. Citation Format: Lydia Greenlees, Li Cheng, Bilal Omar, Yelena Lazdun, Jixin Wang, Fernanda Pilataxi, Michael Kuziora, Zachary Cooper, Luis Vence, Katie Streicher. Blockade of regulatory signaling pathways reduces immunosuppression in the PDAC tumor microenvironment [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 3197.

Abstract 3187: Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome

Abstract Background: KRAS is commonly mutated in solid tumors. Cancer-associated fibroblasts (CAFs) are a key tumor stroma component, which can affect cancer progression and drug response. We hypothesized that defining the differential secreted proteins between CAFs and KRAS mutant-cancer cells could reveal potential mechanisms by which CAFs influence cancer progression and drug resistance. Methods: We studied 12 cell lines: 3 KRAS mutant-colorectal cancer cell lines (H747, LIM2099 and SW620), 3 KRAS mutant-lung cancer cell lines (H1792, H2030 and H23), 3 KRAS mutant-pancreatic cancer cell lines (CAPAN1, DANG, MIAPACA2) and 3 CAF cell line models (colorectal and lung CAF and pancreatic stellate cells). We quantified proteins in cell lines and serum-free media after 24 hour incubation in cells using mass spectrometry in a multiplexed manner (12plex) in triplicates to characterize the proteome and the profile of secreted proteins (secretome), respectively. Prediction software (signalP, secretomeP, TMHMM) and databases (surfaceome and cell surface protein atlas) were used to annotate secreted or transmembrane proteins. Results: Our proteome analysis quantified 9307 proteins. T-test between the cell types (CAF vs cancer) identified 1770 differentially expressed proteins, where 712 proteins were CAF-enriched. This included known CAF markers (e.g α smooth muscle actin) but also proteins not known to be CAF markers such as heat shock protein beta-5/6 (HSPB5/6). The secretome analysis identified 2330 secreted or transmembrane proteins where 202 were differentially expressed between cancer cells and CAFs. 190 proteins were CAF-enriched with the highest ranking being syndecan-2 (SDC2), collagen α-28(I) chain (COL28A1), decorin (DCN) and wnt family member 5B (WNT5B). Interestingly, basal proteome analysis of KRAS mutant-cancer cells showed expression of receptors corresponding to the CAF-enriched ligands, such as receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) which respond to WNT5B. This suggests that CAF-enriched ligands may activate certain downstream pathways, including non-canonical WNT signaling, in KRAS mutant-cancer cells. Conclusion: We undertook deep quantitative proteomics where we identified potential novel CAF markers HSPB5/6. We have also found proteins in the CAF secretome, such as WNT5B, which may drive drug resistance in KRAS mutant-cancer cells. Citation Format: Rachel Lau, Lu Yu, Theodoros Roumeliotis, Lisa Pickard, Adam Stewart, Jyoti Choudhary, Udai Banerji. Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome [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 3187.

Abstract 6125: ADAM9 secreted by cancer-associated stromal cells promotes metastasis of prostate cancer

Abstract Introduction: Cancer-associated fibroblasts (CAFs), the main cellular component of the tumor microenvironment (TME), contribute to tumor metastasis. Prostate cancer (PCa) is the most common malignancy among the elderly male population, and it is typically fatal when the disease progresses to metastasis and castration-resistant stage (CRPC). We have previously demonstrated that ADAM9 increased during PCa progression, and ROS is the critical mediator regulating ADAM9 expression in CRPC cells to promote tumor growth. In addition to membrane-anchored ADAM9, a secreted variant of ADAM9 (sADAM9) was recently identified in activated liver stromal cells that facilitated colon cancer liver metastasis. However, it is not clear whether sADAM9 is expressed by prostatic CAFs and, if so, whether it contributes to aggressive tumor phenotypes through tumor-stromal interactions. Objective: To investigate the sADAM9 variants in CAFs and to elucidate the effects of CAF-derived sADAM9 on the aggressiveness of PCa cells. Methods: Immunohistochemistry staining and ELISA of clinical samples were used to assess the expression of ADAM9 during PCa tumor progression. Laser microdissected tissues, primary cell lines, and experimental cell model of paired normal fibroblasts and CAF cells were analyzed. The effects of sADAM9 on CRPC progression were conducted in PC3 cells by WST-1 cell proliferation assay, transwell migration assay, and μ-slide chemotaxis assay. Immunoblot analyses of integrin β1 and migration-related kinases were used to verify the possible downstream signaling pathway of sADAM9. Results: Increased overall intensity of ADAM9 protein expression in PCa patients with metastasis compared. Elevated ADAM9 expression was observed in both cancerous epithelial cells and the tumor stroma. The average serum ADAM9 concentration measured by ELISA was 6.253 ± 0.448 ng/mL in BPH patient samples versus 10.89 ± 0.953 ng/mL in PCa patient samples. A smaller molecular weight (~50-kDa) of ADAM9 was detected in CM from cultured CAFs but not normal fibroblasts or PCa cell lines except for DU145 cells. Treatment of PC3 cells with sADAM9 induced a significant chemotactic migration while did not affect cell proliferation. Furthermore, a stark increase in activated integrin β1, phosphorylated focal adhesion kinase (p-FAK), and p-AKT, but not p-mTOR in PCa cells by sADAM9 suggested the induction of cell migration occurs through activation of the integrin β1/FAK signaling pathways. Conclusion: Herein, we have provided in vitro evidence to demonstrate the pro-metastatic function of sADAM9 from CAFs that facilitate the migration of CRPC cells through integrin receptor signaling, which could be partly responsible for the promotion of CRPC metastasis. Further molecular identification of controlling spliced ADAM9 expression in CAFs remains to investigate. Citation Format: Weichieh Chen, Le Thi Huynh Trang, Chia-Ling Hsieh, Katsumi Shigemura, Masato Fujisawa, Leland W.K. Chung, Shian-Ying Sung. ADAM9 secreted by cancer-associated stromal cells promotes metastasis of prostate cancer [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 6125.

Abstract 3210: Oncostatin M modulates tumor-fibroblast crosstalk in pancreatic cancer without protecting against cachexia

Abstract Cancer cachexia is the involuntary loss of body weight, skeletal muscle, and adipose tissue associated with disease. Cachexia affects 85% of pancreatic cancer patients and patients suffer the greatest average weight loss among solid cancers. Inflammation is a key driver in cancer cachexia, specifically the Interleukin-6/GP130 family of cytokines that activate signaling of the JAK/STAT pathway. Among family members, Interleukin-6 (IL-6) and Leukemia Inhibitory Factor (LIF) promote cachexia and pancreatic cancer; however, less is known of the related Oncostatin M. Here we show cachexia and elevated circulating Osm coincide in the KrasG12D;Trp53R172H;Pdx1:Cre genetically engineered mouse model (KPC-GEMM) of pancreatic cancer and in mice bearing KPC cells implanted orthotopically. Osm expression was elevated in cardiac muscle and adipose tissue in KPC cachexia, while OSM receptor (Osmr) was elevated in cardiac and skeletal muscle and liver, indicating a systemic OSM circuit. Consistent with a feed-forward loop, KPC cells treated with OSM upregulated expression of OSMR. RNA-sequencing of OSM treated KPC and cancer-associated fibroblast (CAF) cell lines revealed activation of inflammation, fibrosis, and tumor progression pathways, including induction of IL-6. Functionally, exogenous OSM caused cardiac dysfunction, local muscle atrophy, fibrosis, and systemic bone loss in wildtype and Il6 null mice, suggesting OSM might mediate cachexia independent of IL-6. We hypothesized that deletion of OSM would be protective in cancer cachexia. Thus 14-week-old WT and Osm null mice were orthotopically implanted with KPC cells or underwent sham surgery; all groups were euthanized when one reached 5% fat mass. KPC tumors caused muscle weakness, body deconditioning and reduced activity as well as anemia and hematological inflammation. These findings were not genotype specific. There were also no differences in KPC-induced cardiac, skeletal muscle or fat wasting, nor in splenomegaly, organ wasting, or tumor size between WT and Osm null mice. Similarly, Osmr null mice showed no differences in KPC cachexia phenotypes versus wildtype mice. Nevertheless, tumors from Osm null mice showed alterations in the stromal compartment, including reduction in collagens I and III, decreased alpha smooth muscle actin staining, and altered mitochondrial metabolism and cellular composition by proteomics and RNAseq. Consistent with a role for OSM in modulating the stroma, excess OSM induced compaction of tumor cells and CAFs in 3D co-cultures. Together these data indicate that OSM signaling influences tumor-fibroblast crosstalk in the pancreatic cancer microenvironment but is not necessary for cachexia in the macroenvironment. Citation Format: Daenique H. Jengelley, Ashok Narasimhan, Joseph E. Rupert, Xiaoling Zhong, Andrew R. Young, Leonidas G. Koniaris, Teresa A. Zimmers. Oncostatin M modulates tumor-fibroblast crosstalk in pancreatic cancer without protecting against cachexia [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 3210.

Abstract 269: PD-L1 dominated tumor-CAF-immune cell crosstalk of stromal microenvironment: Endometrial tumor vs. tumor-adjacent normal

Abstract Background: In endometrial cancers (EC), the mechanistic contribution of PD-L1/L2 and PD-1 signaling of host’s stromal microenvironment (SME) [cancer-associated fibroblast (CAF) and immune cells] in relation to the tumor (T) cells is elusive. Aim: To understand the tumor-stroma-immune crosstalk in EC, we studied the compartmental pattern of PD-L1, PD-L2, and PD-1 expression in EC tissues and their matched CAFs from both T and tumor-adjacent normal (N) tissues. Method: Over 116 surgically resected T and N tissues were obtained from consented patients with EC at the Avera Cancer Institute (March ‘16 and September ’21). IHC expression was performed in T, N-epithelium, SME (mesenchyme) in the T and N tissues. The staining intensity and distribution pattern were evaluated by a pathologist using a standard scoring system (1% cut-off value). Since fibroblasts are the most abundant cells of SME, the PD-L1 was parallelly evaluated in the multiple passages of cultured T and N derived CAFs by the qRT-PCR, flow-cytometry, and ICC. Result: The expression of 3 immune checkpoint proteins in the epithelial and mesenchyme of N was limited to only rare and focal positivity for PD-L1. The epithelial compartments of the T tissue, in contrast, had 70% positivity for PD-L1, which was independent of differentiation status, presence of TILs, or lymphovascular invasion. PD-L1 positivity was 90% of the SME of tested samples, identified predominantly in the (1) immune component, including macrophages and lymphocytes, and (2) EpCAM-/SMA+/FAP+/S100A4+ CAFs. Both PD-L1 and PD-L2 were identified by flow, ICC, and qRT-PCR in NCAF and TCAF. The overall expression of PD-L2 was found to be qualitatively and quantitatively lesser than that of PD-L1, primarily contributed by SME/stromal macrophages and lymphocytes. PD-1 expression was restricted to the SME/stromal immune components, predominantly lymphocytes. The expression for PD-L1 and PD-L2 mRNA was higher in TCAF than NCAF in successive passages, which ICC and flow cytometry confirmed. Summary: The expression of immune checkpoint proteins, PD-L1, and PD-L2, are primarily tumor-driven and associated with the endometrioid (invasive) and serous adenocarcinomas of grades II and above with myometrial invasion. The expression of PD-L1 and PD-L2 are distributed in both tumor and SME/stromal components, while PD-1 expression is exclusively SME/stromal. Citation Format: Xiaoqian Lin, Jennifer Aske, Adam Dale, Nischal Koirala, Ethan Thompson, Mary Fagerness, Natasha Flier, Paige Davies, Stacy Kehoe, Cheryl Ageton, Kris Gaster, Luis Rojas Espaillat, David Starks, Raed Sulaiman, Pradip De, Nandini Dey. PD-L1 dominated tumor-CAF-immune cell crosstalk of stromal microenvironment: Endometrial tumor vs. tumor-adjacent normal [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 269.

Abstract 1565: Targeting stromal-specific p38 MAPK signaling to stifle inflammatory reprogramming of cancer-associated fibroblasts in pancreatic cancer

Abstract Introduction: Major contributors to therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) are the desmoplastic stroma that acts as a barrier to drug delivery and effector immune cell infiltration. In our efforts to identify mediators of therapeutic resistance, we identified the pro-inflammatory cytokine interleukin-1α (IL-1) to be critical in the activation and reprogramming of neighboring cancer-associated fibroblasts (CAFs) within the stroma to a pro-inflammatory phenotype to promote myeloid cell chemotaxis. Our mechanistic studies have identified the p38 MAPK pathway as a novel mediator of IL-1-induced CAF activation from quiescent stellate cells into the inflammatory fibroblast. Our central hypothesis is that disruption of the IL-1/p38 MAPK signaling cascade in pancreatic stellate cells (PSCs) and CAFs can improve therapeutic resistance by remodeling the fibrotic stromal landscape and the overall immune microenvironment in PDAC tumors. Methods: Inhibition of phosphorylated p38 MAPK was achieved pharmacologically with Pexmetinib and genetically with an shRNA lentiviral system in CAF and PSC cell lines. Inflammatory PSC/CAF activation was determined by qPCR, immunofluorescence, and vitamin A assay. Ptf1acre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with the p38 inhibitor, pexmetinib (30mg/kg, daily PO), or vehicle control for 2.5 weeks prior to sacrifice. For survival studies, PKT mice were treated with vehicle, gemcitabine (20 μg/twice weekly), pexmetinib, or combination until moribund. Results: Inhibition of p38 MAPK in PSCs prevented activation into an inflammatory fibroblast in vitro when stimulated with IL-1 or tumor cell cocultures. p38 MAPK inhibition in vivo resulted in signification reduction of PDGFR+ CAFs in a PDAC GEMM. Additionally, p38 MAPK inhibition resulted in a significant reduction of circulating myeloid cells (CD11b+) and intratumoral monocytic MDSCs (Ly6C+). p38 inhibition, in combination with chemotherapy, significantly improves overall survival in a PDAC GEMM. Conclusions: These findings provide important mechanistic data to explore p38 MAPK inhibition to target the fibrotic stroma and reduce immunosuppressive myeloid levels in tumors and provide compelling preclinical evidence to combine pexmetinib with chemotherapy to improve overall survival in PDAC. Citation Format: Samara Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Anna Bianchi, Vanessa Tonin Garrido, Nilesh Deshpande, Zhiqun Zhou, Jashodeep Datta, Nagaraj Nagathihalli, Nipun Merchant. Targeting stromal-specific p38 MAPK signaling to stifle inflammatory reprogramming of cancer-associated fibroblasts in pancreatic cancer [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 1565.

Abstract 3195: Tumor inhibitory CAFs in CNS metastases

Abstract The 2-year survival rate for patients with central nervous system (CNS) metastases (CMs) remains below 2%. With the aim to define new therapeutic options, researchers have investigated how interactions between CM cells and the surrounding tumor microenvironment (TME) support metastatic progression. As an important component of the TME, cancer associated fibroblasts (CAFs) have largely been reported as tumor supportive. But new evidence for the high heterogeneity of the CAF compartment, including in vivo studies from our lab, showed that certain CAF subpopulations could exhibit tumor inhibitory capabilities. Interestingly, recent reports revealed that CAFs showing high expression of Immunoglobulin Superfamily containing Leucin Rich Repeat (ISLR) could show tumor inhibitory capabilities in both pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). Yet, despite the invaluable clinical potential of tumor inhibitory CAFs, the CAF compartment in the TME of CMs is still largely under described. Using CM patient tissue and CM patient-derived cell lines established in our lab, we performed single cell RNA sequencing (scRNA-seq) to define CAF subpopulations present in the TME of CMs. We identified distinct clusters characterized by differential expression of ISLR and tumor supportive CAF marker Actin Alpha 2, Smooth Muscle (ACTA2/α-SMA). CAF subpopulations were thus defined as follows: ISLRhigh/α-SMAlow, ISLRlow/α-SMAhigh, ISLRlow/α-SMAlow. Altogether, we hypothesized that ISLRhigh/α-SMAlow CAFs could act to restrain tumor progression in the TME of CMs, as opposed to potentially tumor supportive ISLRlow/α-SMAhighCAFs. ISLRhigh and ISLRlow CAFs were obtained through fluorescence-activated cell sorting (FACS) performed on one of our patient-derived CAF cell lines represented in all defined scRNA-seq clusters. ISLRhigh CAFs were validated to express lower levels of α-SMA (ISLRhigh/α-SMAlow CAFs) compared to ISLRlow cells (ISLRlow/α-SMAhigh CAFs). This mutually exclusive expression of ISLR and α-SMA in different CAF subpopulations is in accordance with recent data in PDAC and CRC and could indicate common underlying mechanisms driving the phenotype of each subpopulation. Further in vitro and in vivo experiments are currently underway in our lab to determine the tumor supportive capabilities of both ISLRhigh/α-SMAlow and ISLRlow/α-SMAhigh CAFs. Ultimately, we expect the current study to unravel the diverse and dynamic nature of CAFs in the TME of CMs, which is still largely unknown. Citation Format: Thomas Simon, David Buckley, Bodour Salhia. Tumor inhibitory CAFs in CNS metastases [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 3195.

Abstract 3646: Distinct functions of pancreatic stellate cells in PDAC stromal evolution

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers in the United States with a five-year relative survival rate of 10.8%. After surgery, radiation and chemotherapy are the next lines of therapy. However, these treatments have not significantly improved clinical outcomes due in part to its unique and characteristic desmoplasia, which limits treatment efficacy. Cancer-associated fibroblasts (CAFs) have been shown to contribute in PDAC’s stromal reaction in part viasecretion of extracellular matrix (ECM) components and growth factors, suggesting that CAFs are pro-tumorigenic. This understanding led to several CAF ablation preclinical studies; surprisingly, these studies demonstrated tumor-suppressive potential for CAFs, highlighting the functional heterogeneity among CAFs in PDAC. This emphasizes the need for greater developmental understanding of this influential cell population. Lineage tracing via our recently established Fabp4-Cre;Rosa26mTmG mouse model revealed that pancreatic stellate cells (PSCs), thought to be the main cellular origin of PDAC CAFs, contribute to a minor faction of CAFs yet appear to have non-redundant functions to alternatively-derived CAFs. Here we delve further into the specific functions of PSC-derived CAFs via single-cell RNA sequencing of pancreatic stellate cells in healthy and tumor pancreas with our Fabp4-Cre;Rosa26mTmG mouse model. To our surprise, initial scRNA-seq analysis illustrated that quiescent pancreatic stellate cells from healthy tissue appear to be more heterogeneous to PSC-derived CAFs, with quiescent pancreatic stellate cells clustering into three “super clusters” while PSC-derived CAFs clusters into one “super cluster”. Additionally, clusters specific to normal pancreas tissue revealed potential transcripts-of-interest that are anti-tumorigenic. Current efforts seek to analyze stromal evolution with respect to CAF cell of origin in pre-malignant lesions and throughout stepwise tumorigenesis to metastasis. Furthermore, we are investigating the functional significance of potentially anti-tumorigenic factors lost from the PSC compartment upon differentiation to a CAF phenotype during PDAC progression. These findings shed light on the increasingly appreciated heterogeneity of PDAC CAFs, whilst suggesting that PSC-derived CAFs may be specifically targeted as a singular cell population for therapeutic treatment. Importantly, these findings help gain a greater understanding of PDAC disease progression and development in order to better serve patient care. Citation Format: M. Kathrina Casapao Onate, Matthew Schleisman, Zheng Xia, Mara H. Sherman. Distinct functions of pancreatic stellate cells in PDAC stromal evolution [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 3646.

Abstract 1572: FZD6 and RYK, non-canonical Wnt receptors in pancreatic cancer progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a grim prognosis with 5-year patient survival of less than 10%. Development of effective therapeutics has been hampered by the PDAC tumor microenvironment (TME), which contains dense desmoplastic stroma that likely impedes the ability of therapies to reach the tumor cells. We have found that aggressive, “basal-like” human PDAC tumors have high expression of proteins involved in planar-cell-polarity (PCP), a type of non-canonical Wnt signaling. We show that the increased expression of the PCP genes, including the putative non-canonical Wnt ligand, Wnt5a, the Frizzled 6 (FZD6) Wnt receptor, and RYK co-receptor negatively impact patient prognosis. Using a panel of FZD6 knock-down pancreatic cell lines, we show that loss of FZD6 increases expression of the epithelial marker, E-cadherin, and decreases cellular motility in Transwell assays. We also find that loss of Fzd6 dramatically inhibits tumor progression in two mouse models of PDAC, with a pronounced change to the TME; including the cancer-associated fibroblasts (CAFs) and immune cells. Blinded histologic pathology review of tumor and immune cell infiltrates, by H&E and alphaSMA staining, suggests that the CAF cell population is altered. We propose this is the cause of the looser stroma surrounding the tumors after Fzd6 loss, which also results in an increased abundance of CD4 and CD8 positive T cells surrounding and within the tumors. Additionally, immunohistochemical staining for other PCP proteins within the pancreatic tumors lacking Fzd6 suggests that Fzd6 loss reduces PCP-component protein levels. We hypothesize that the loss of Fzd6 reduces tumor cell EMT and PCP signaling, while the loose stroma allows more immune cells that further slow tumor growth and progression. We have also examined the expression of PCP proteins in a novel orthotopic xenotransplantation model of PDAC, intraductally grafted organoids (IGO). IGO is a powerful tool to study PDAC as the intraductal tumors that develop recapitulate patient to patient heterogeneity while displaying features of either classical or basal-like PDAC subtypes. FZD6 and RYK are both more highly expressed in the IGO basal-like tumors than the classical type. Introduction of fast-growing, basal-like human tumors into the murine pancreas also resulted in a TME that produces more Wnt5a, when compared to the stroma surrounding tumors formed by injecting the classical subtype. Taken together, we believe we have identified a Wnt5a/Fzd6 driven PCP-like signaling pathway that makes tumors more mesenchymal-like, and results in PDAC tumor progression and worse patient prognosis. Citation Format: Payton D. Stevens, Emily Sall, Alex Zhong, Galen Hostetter, Zachary Madaj, Jennifer Thalappillil, Alexander Dobin, Youngkyu Park, David A. Tuveson, Bart O. Williams. FZD6 and RYK, non-canonical Wnt receptors in pancreatic cancer progression [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 1572.

Immune Subtypes in LUAD Identify Novel Tumor Microenvironment Profiles With Prognostic and Therapeutic Implications.

The six transcriptomic immune subtypes (ISs) (C1 - C6) were reported to have complex and different interplay between TME and cancer cells in TCGA (The Cancer Genome Atlas) pan-cancer cohort. Our study specifically explored how the consequence of interplay determines the prognosis and the response to therapy in LUAD cohorts. Clinical and molecular information of LUAD patients were from TCGA and Gene Expression Omnibus (GEO). The immune cell populations and gene/pathway enrichment analysis were performed to explore the molecular differences among the C3 IS and other ISs in the LUAD population. The proportion of C3 inflammatory IS was identified as the most common IS in both TCGA (N = 457) and GEO (N = 901) cohorts. The C3 IS was also found to be the most accurate prognostic subtype, which was associated with significantly longer OS (p <0.001) and DFS (p <0.001). The C3 IS presented higher levels of CD8 T, M1 macrophage, and myeloid dendritic cells, while lower levels of M2 macrophages and cancer-associated fibroblast cells. Moreover, the C3 subtype was enriched in the antigen process and presenting, interferon-gamma response, T cell receptor signaling, and natural killer cell-mediated cytotoxicity pathways than C1/C2. In contrast, the C1/C2 presented greater activation of pathways related to the cell cycles, DNA repair, and p53 signaling pathways. The immune-related C3 IS had a great ability to stratify the prognosis of LUAD, providing clues for further pathogenic research. This classification might help direct precision medicine screenings of LUAD patients, thus possibly improving their prognoses.

Signal peptidase complex catalytic subunit SEC11A upregulation is a biomarker of poor prognosis in patients with head and neck squamous cell carcinoma.

In the current study, we aimed to investigate the expression of the five microsomal signal peptidase complex (SPC) subunit genes (SEC11A, SEC11C, SPCS1, SPCS2, and SPCS3) in head and neck squamous cell carcinoma (HNSC) and to explore their prognostic value. Data from the HNSC subset of The Cancer Genome Atlas (TCGA) and one previous single-cell RNA-seq dataset was used. Subgroup analysis was conducted in tumors from different anatomic sites. Gene set enrichment analysis (GSEA), and immune cell infiltration analysis were performed to check the influence of SEC11A on the tumor microenvironment. Among the genes significantly upregulated in the tumor group, only SEC11A expression (as a continuous variable) is independently associated with poorer progression-free survival (PFS) (HR: 2.075, 95%CI: 1.447–2.977, p<0.001) and disease-specific survival (DSS) (HR: 2.023, 95%CI: 1.284–3.187, p = 0.002). Subgroup analysis confirmed the prognostic value in tumors from three anatomic origins, including laryngeal squamous cell carcinoma, oral cavity-related squamous cell carcinoma, and oropharynx-related squamous cell carcinoma. SEC11A is expressed in all subtypes of cells in the tumor microenvironment. Its expression showed a moderate positive correlation with its gene-level copy number (Pearson’s r = 0.53, p<0.001). SEC11A expression was negatively correlated with CD8+ T cells and B cells, but was positively correlated with cancer-associated fibroblast and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. In summary, SEC11A upregulation is a result of gene amplification in head and neck squamous cell carcinoma. Its upregulation might serve as an independent prognostic biomarker and a predictor of the infiltration of certain types of immune cells.

Integrin-Linked Kinase Expression Characterizes the Immunosuppressive Tumor Microenvironment in Colorectal Cancer and Regulates PD-L1 Expression and Immune Cell Cytotoxicity.

Integrin-linked kinase (ILK) has been implicated as a molecular driver and mediator in both inflammation and tumorigenesis of the colon. However, a role for ILK in the tumor microenvironment (TME) and immune evasion has not been investigated. Here, we show a correlation of ILK expression with the immunosuppressive TME and cancer prognosis. We also uncover a role for ILK in the regulation of programmed death-ligand 1 (PD-L1) expression and immune cell cytotoxicity. Interrogation of web-based data-mining platforms, showed upregulation of ILK expression in tumors and adjacent-non tumor tissue of colorectal cancer (CRC) associated with poor survival and advanced stages. ILK expression was correlated with cancer-associated fibroblast (CAFs) and immunosuppressive cell infiltration including regulatory T cells (Treg) and M2 macrophages (M2) in addition to their gene markers. ILK expression was also significantly correlated with the expression of different cytokines and chemokines. ILK expression showed pronounced association with different important immune checkpoints including PD-L1. Deletion of the ILK gene in PD-L1 positive CRC cell lines using a doxycycline inducible-CRISPR/Cas9, resulted in suppression of both the basal and IFNγ-induced PD-L1 expression via downregulating NF-κB p65. This subsequently sensitized the CRC cells to NK92 immune cell cytotoxicity. These findings suggest that ILK can be used as a biomarker for prognosis and immune cell infiltration in colon cancer. Moreover, ILK could provide a therapeutic target to prevent immune evasion mediated by the expression of PD-L1.

Tumorigenic and immunological roles of Heat shock protein A2 in pancreatic cancer: a bioinformatics analysis.

Heat shock protein A2 has been reported to be tightly associated with tumorigenesis and tumor progression. This study aimed to determine the oncogenic and immunological roles of Heat shock protein A2 in pancreatic cancer by bioinformatics. Expression of Heat shock protein A2 in tumorous and normal specimens of pancreatic cancer was analyzed using the Cancer Genome Atlas and the Cancer Genome Atlas + Genotype-Tissue Expression data sets, respectively. Relationships of Heat shock protein A2 expression with immune infiltrates in pancreatic cancer were assessed. Heat shock protein A2-associated coexpressed genes in pancreatic cancer were obtained, followed by the implementation of enrichment analysis. The data demonstrated that Heat shock protein A2 was significantly overexpressed in tumorous samples compared with normal samples. Heat shock protein A2 expression was remarkably positively interrelated with CD8+ T cell, neutrophil, dendritic cell, and macrophage, but not with CD4+ T and B cells. Heat shock protein A2 expression was markedly positively relevant to both cancer-associated fibroblast and endothelial cell. Enrichment data revealed that Heat shock protein A2 was intimately involved in the tumorigenesis and progression of pancreatic cancer. Heat shock protein A2 is upregulated in pancreatic cancer and is closely associated with tumor immunity and aggressive progression.

Cytokine-driven positive feedback loop organizes fibroblast transformation and facilitates gastric cancer progression.

Gastric cancer (GC) is a malignancy that belongs to one of the most common leading causes of cancer death. Cancer-associated fibroblasts (CAFs) promote the GC cells' malignant behavior. It is still unknown how GC converts normal fibroblasts (NFs) to CAFs. GC cells were co-cultured with NFs. Bioinformatics was used to analyze the genes and signaling pathways that were changed in fibroblast. RT-PCR, western blot, and Elisa assays were used to detect the expression of cytokines in fibroblast and condition medium. Western blot and immunofluorescence demonstrated activation of relevant pathways in CAFs-like cells. Transwell, scrape, colony formation, and CCK-8 assays were performed to reveal the feedback effect of CAFs-like cells on GC cells. GC promoted the conversion of NFs to CAFs by secreting Interleukin 17A (IL-17). It included both morphological and molecular marker changes. This process was achieved by activating the nuclear factor-κB (NF-κB) pathway. On the other hand, CAFs cells could secrete C-X-C Motif Chemokine Ligand 8 (IL-8, IL-8), which promoted the malignant phenotype of GC cells. In this way, a feedback loop of mutual influence was constructed in the GC and tumor microenvironment (TME). Our research proved a novel model of GC-educated NFs. GC-IL-17-fibroblast-IL-8-GC axis might be a potential pathway of the interaction between GC and TME.

Abstract P5-06-08: Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis

Abstract Background: Metastatic disease is the foremost cause of breast cancer (BC) related mortality in women. One of the crucial challenges in reducing metastasis-related mortality is in identifying and understanding why certain BCs metastasize and recur. Although cancer cell-intrinsic factors are a key determinant in BC metastasis, host-intrinsic factors like the cells of the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs) are what might be driving certain BCs to metastasize and recur. We recently showed that CAFs from the primary (TME) enter into circulation as cCAFs, form heterotypic clusters with tumor cells, and arrive at metastatic sites. This interaction between CAFs and tumor cells is crucial in furthering the metastatic cascade. However, the functional heterogeneity of the different CAF phenotypes and how that impacts BC metastasis is unknown. Stromal derived factor-1 (SDF-1/CXCL12) is an important chemokine that is known to be involved in promoting tumor cell invasion and metastasis. In this study, we examine the functional differences between CAF isolated from different molecular subtypes of BC. Furthermore, we elucidate the role of CAF secreted SDF-1 as being one of the mechanisms by which a subset of CAF cells permanently reprograms poorly-metastatic MCF-7 cells to augment EMT-driven genes and become metastatic in vivo. Methods: We used primary CAF cell lines derived from BCs of different molecular subtypes and examined their global gene expression profiles using RNAseq. We used NSG mice xenografted with MCF-7 BC cells and primary CAF cells to evaluate the contribution of two different CAF phenotypes in promoting BC progression and metastasis. We developed dissociated tumor cell lines from CAF co-injected MCF-7 xenografts to determine the molecular changes that CAFs impart to MCF-7 cells in vivo. Using limiting dilution tumor xenograft assays and gene expression profiles we examined the molecular pathways, BC stemness markers, and tumor-initiating capacity of the dissociated tumor cell lines. By long-term SDF-1 treatment of MCF-7 cells in vitro and using these cells in various RNASeq, in vitro, and in vivo assays we determined the role of SDF-1 in altering the phenotype of MCF-7 cells. Results: We found that CAFs from different molecular subtypes have a differential effect on tumor initiation, progression, and metastasis in xenograft assays. We also found that a subset of CAFs has the ability to make the poorly-metastatic MCF-7 cells metastatic in vivo. We also found that this subset of CAF cells has a higher expression of SDF-1 and the tumors formed with the co-injection of MCF-7 cells with this subset of CAF cells are enriched in cancer stem cell-like metastasis initiating cells. We also found that upon serial transplant, the tumors formed by CAF-reprogramed MCF-7 cells have a gene expression profile that shows enrichment for EMT-driven genes and these tumors are robustly metastatic. Furthermore, we found that the SDF-1/CXCR4 axis is one of the critical mechanisms by which this subset of CAF cells permanently reprograms the phenotype of the MCF-7 cells. Conclusions: CAFs are a highly heterogeneous population of cells in the breast TME. This study examines the contribution of the functional heterogeneity of the CAF cells in BC metastasis. We have found that a unique subset of CAF cells has the ability to reprogram a poorly metastatic BC cell to not only become metastatic but induce a permanent EMT-driven phenotypic shift in the BC cells. SDF-1 may be one of the mechanisms that drive this CAF-induced change and understanding the CAF functional landscape and its role in BC metastasis might be crucial to developing new therapeutic modalities to abrogate BC metastasis. Citation Format: Utsav Sharma, Jun Sun, Kelsie Medina-Saenz, Susan Bare, Philip Miller, Manuel Picon-Ruiz, Joyce Slingerland, Dorraya El-Ashry, Marc Lippman. Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-08.