The immunologic and stromal functions of nitric oxide in fibrotic tumor microenvironments: A mini-review

Cancer-metastasis-on-a-chip reveals efficacy of bevacizumab and siRNA in overcoming carboplatin resistance in SKOV3 ovarian cancer within a fibrotic metastatic microenvironment

Pancreatic cancer is not responsive to targeted therapy. This may be due to the presence of a uniquely fibrotic and immunosuppressive tumor microenvironment present in pancreatic ductal adenocarcinoma (PDAC). Critical obstacles to targeted therapy in PDAC tumors include the dense desmoplastic stroma that acts as a physical barrier to drug delivery and the high numbers of tumor‐associated immunosuppressive cells. In our previous study, we identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive tumor microenvironment (TME). FAK inhibition (VS‐4718) significantly limited tumor progression, and prolonged mice survival. Herein, we observed that STAT3 signaling was constantly activated in non‐responsive and recurrent tumors, suggesting STAT3 signaling pathway regulates FAK inhibitor (FAKi) response and resistance. Furthermore, gene enrichment analysis showed that TGF‐b signaling was significantly downregulated upon FAK inhibition, suggesting a negative feed back regulation of STAT3 by TGF‐ b signaling. We proposed that overcoming STAT3 reactivation upon FAK inhibition would enhance pancreatic cancer sensitive to FAK inhibitor. Accordingly, we found that combined STAT3 inhibition (Stattic) with FAK inhibition significantly prolonged the survival in the p48‐Cre/LSL‐KrasG12D/p53Flox/ Flox (KPPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of immunosuppressive myeloid cells and increased tumor cell death. Together, our data indicate that STAT3 inhibition sensitizes PADC to FAKi and overcomes FAKi resistance.Support or Funding InformationThis work was supported by Lustgarten Foundation, an AACR/PANCAN Award, NCI awards R01‐CA177670, R01‐CA203890 and R21‐CA182701This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The arachidonic acid cascade is a major inflammatory pathway that produces prostaglandin E2 (PGE2). Although inhibition of 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) is reported to lead to PGE2 accumulation, the role of 15‐PGDH expression in the tumor microenvironment remains unclear. We utilized Panc02 murine pancreatic cancer cells for orthotopic transplantation into wild‐type and 15‐pgdh+/− mice and found that 15‐pgdh depletion in the tumor microenvironment leads to enhanced tumorigenesis accompanied by an increase in cancer‐associated fibroblasts (CAFs) and the promotion of fibrosis. The fibrotic tumor microenvironment is widely considered to be hypovascular; however, we found that the angiogenesis level is maintained in 15‐pgdh+/− mice, and these changes were also observed in a genetically engineered PDAC mouse model. Further confirmation revealed that fibroblast growth factor 1 (FGF1) is secreted by pancreatic cancer cells after PGE2 stimulation, consequently promoting CAF proliferation and vascular endothelial growth factor A (VEGFA) expression in the tumor microenvironment. Finally, in 15‐pgdh+/−Acta2‐TK mice, depletion of fibroblasts inhibited angiogenesis and cancer cell viability in orthotopically transplanted tumors. These findings highlighted the role of 15‐pgdh downregulation in enhancing PGE2 accumulation in the pancreatic tumor microenvironment and in subsequently maintaining the angiogenesis level in fibrotic tumors along with CAF expansion.

Fibrotic immune microenvironment remodeling mediates superior anti-tumor efficacy of a nano-PD-L1 trap in hepatocellular carcinoma

Steatotic liver enhances liver metastasis of colorectal cancer (CRC), but this process is not fully understood. Steatotic liver induced by a high-fat diet increases cancer-associated fibroblast (CAF) infiltration and collagen and hyaluronic acid (HA) production. We investigated the role of HA synthase 2 (HAS2) in the fibrotic tumor microenvironment in steatotic liver using Has2ΔHSC mice, in which Has2 is deleted from hepatic stellate cells. Has2ΔHSC mice had reduced steatotic liver-associated metastatic tumor growth of MC38 CRC cells, collagen and HA deposition, and CAF and M2 macrophage infiltration. We found that low-molecular weight HA activates Yes-associated protein (YAP) in cancer cells, which then releases connective tissue growth factor to further activate CAFs for HAS2 expression. Single-cell analyses revealed a link between CAF-derived HAS2 and M2 macrophages and CRC cells through CD44; these cells were associated with exhausted CD8+ T cells via programmed death-ligand 1 and programmed cell death protein 1 (PD-1). HA synthesis inhibitors reduced steatotic liver-associated metastasis of CRC, YAP expression, and CAF and M2 macrophage infiltration, and improved response to anti-PD-1 antibody. In conclusion, steatotic liver modulates a fibrotic tumor microenvironment to enhance metastatic cancer activity through a bidirectional regulation between CAFs and metastatic tumors, enhancing the metastatic potential of CRC in the liver.

Who am I? (re-)Defining fibroblast identity and immunological function in the age of bioinformatics.

Tumor microenvironment consists of tumor cells, stromal cells, extracellular matrix and a plethora of soluble components. The complex array of interactions between tumor cells and their surrounding tumor microenvironments contribute to the determination of the fate of tumor cells during tumorigenesis and metastasis. Matricellular protein periostin is generally absent in most adult tissues but is highly expressed in tumor microenvironments. Current evidence reveals that periostin plays a critical role in establishing and remodeling tumor microenvironments such as the metastatic niche, cancer stem cell niche, perivascular niche, pre-metastatic niche, fibrotic microenvironment and bone marrow microenvironment. Here, we summarize the current knowledge of the multifaceted role of periostin in the tumor microenvironments.

Recent findings indicate that in cancer, host immune suppression and resistance to therapy is orchestrated by the presence and activity of stromal cells in the tumor microenvironment. Therefore novel approaches to overcome stromal mediated resistance to therapy represent a potential strategy to improve the outcome to chemotherapy and immune checkpoint blockade, particularly in tumors that have a fibrotic microenviroment. Autotaxin is an enzyme that converts Lysophosphatidylcholine (LPC) to Lysophosphatidic Acid (LPA), a key pathway that is aberrantly activated in fibrosis. LPA has also been described to act directly on tumor cells, promoting their growth and proliferation via G protein-coupled LPA Receptors (LPARs). IOA-289 is a novel, potent, selective and orally bioavailable inhibitor of Autotaxin, a target with known clinical application in fibrotic diseases and with a strong rationale for exploration in cancer. In preclinical models, IOA-289 modulated levels of circulating LPA in a dose dependent manner. At doses achieving at least 50% reduction of circulating LPA, IOA-289 showed monotherapeutic efficacy in in vivo mouse models of cancer. Notably the effects were most prominent in orthotopic models that recapitulate the stromal components of the microenvironment. In vitro studies investigated the mechanism of action of IOA-289, and it was shown to be driven by: (i)Direct anti-tumor activity on cancer cell lines in vitro (ii)Modulation of the secretory profile of fibroblasts(iii)Increased T cell infiltration in vitro and in vivo In a healthy volunteer study following a single oral ascending dose, IOA-289 showed a dose dependent increase in plasma and a corresponding decrease in circulating LPA. We also demonstrated that ATX was elevated in plasma from pancreatic cancer patients compared to samples from healthy volunteers and are correlated with soluble CA19-9. Based on our preclinical data we postulate that inhibition of the ATX/LPA pathway with IOA-289 may be a beneficial therapeutic strategy for cancer patients with fibrotic tumor microenvironments. The predicted biologically effective dose of IOA-289 has been calculated using PK/PD modelling combining data from preclinical studies and from the phase Ia healthy volunteer study. IOA-289 is scheduled to enter a phase Ib clinical trial in pancreatic cancer. Citation Format: Zoe Johnson, Marcel Deken, Ragini Medhi, Lauren Maggs, Alan Carruthers, Anne Cheasty, Alessia Tagliavini, Andrea Nizzardo, Marco Pergher, Karolina Niewola, Amy Fraser, Lars van der Veen, Pritom Shah, Luigi ZIviani, Stefano Milleri, Michael Lahn. Targeting Autotaxin to suppress stromal signaling in the tumor microenvironment to improve outcome to therapy in fibrotic tumor types [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 2636.

A fibrotic tumor microenvironment (TME) promotes tumor progression by the interactions between tumor cells and cancer-associated fibroblasts (CAFs) in the extracellular matrix (ECM), which enhance tumor cell survival and growth, and by suppressing antitumor immunity. However, how characteristic gene expression and amplification in cancer cells drive the formation of a fibrotic TME in patients with gastric cancer (GC) is unclear. We performed genomic and transcriptomic analyses via datasets from The Cancer Genome Atlas and the Asian Cancer Research Group to identify amplified and overexpressed genes associated with the presence of a fibrotic TME in tumors. Syngeneic mouse models of GC and multiplexed immunohistochemistry (IHC) were used to validate the findings of the transcriptomic analysis and investigate the underlying mechanisms. The Frizzled class receptor 1 (FZD1) gene was frequently amplified and highly expressed in GC patients with fibrotic tumors, and FZD1 expression was related to a poor prognosis. The overexpression of Fzd1 in murine GC tumor cells was significantly associated with enhanced tumor fibrosis and growth and reduced infiltration of CD3+ lymphocytes and CD8+ T cells into tumors. SLIT2 secretion was increased in Fzd1-overexpressing tumor cells via the canonical WNT-β-catenin pathway, and SLIT2 activated CAFs to produce more ECM through the SLIT2‒ROBO1 axis. This study highlights the potential of FZD1 as a biomarker for predicting fibrotic status in patients with GC and the SLIT2‒ROBO1 axis as a therapeutic target to reverse a fibrotic and immunosuppressive TME.

Grapefruit extracellular vesicle (GEVs) derived nanodrug Tf-GEVs@Pt(IV) loading novel three-functional platinum(IV) conjugates was developed, which was effective in modulating the inflammatory, fibrotic, and immunosuppressive tumor microenvironment (TME). The GEVs enhanced the penetration of Tf-GEVs@Pt(IV) into tumor tissues, while the transferrin (Tf) moiety further improved the tumor-targeting capabilities. The active ingredient, platinum(IV) conjugate, was released in a sustained manner within the TME, which was easily reduced to platinum(II), aspirin (ASP), and captopril (CTP) fragments. The platinum core caused significant DNA damage. The inflammatory TME was ameliorated by inhibiting COX-2 and reducing TNF-α and IL-6 through the synergistic actions of GEVs and ASP. Collagen fibrosis was disrupted by the CTP moiety, which downregulated TGF-β1, MMP2, and MMP9. Consequently, the immunologically 'cold' TME was transformed into a 'hot' state by inhibiting PD-L1 and activating cGAS/STING pathways. Then, the T-cell activation and macrophage polarization from the M2- to M1-phenotype were provoked in tumor tissues.

High-grade serous ovarian carcinoma (HGSOC), is associated with high mortality rates due to late-stage diagnosis and limited treatment options. We investigated the role of FSTL3 in ovarian cancer progression both as a prognostic biomarker and as a potential therapeutic target.We measured levels of follistatin (FST) and follistatin-like 3 (FSTL3) in 96 ovarian cancer patient ascites samples and found that FSTL3 overexpression was more predominant than FST and associated with poorer survival outcomes. Mice implanted with an HGSOC syngeneic cell line bearing common alterations in ovarian cancer (KRASG12V, P53R172H, CCNE1oe, AKT2oe) had increasing levels of FST and FSTL3 in serum during tumor growth. Further alteration of this model to generate a knockout of FST (KPCA.FSTKO) and an overexpression of human FSTL3 (KPCA.FSTKO_hFSTL3), revealed that FSTL3 expression was associated with a more fibrotic tumor microenvironment, correlating with an increased abundance of cancer-associated myofibroblasts (myCAFs), and cancer cells with a more mesenchymal phenotype. Tumors overexpressing FSTL3 had less immunocyte infiltration and a significantly reduced intratumoral T-cell abundance (CD4+, CD8+). FSTL3 overexpression completely abrogated tumor response to PPC treatment (Prexasertib combined with PD-1 and CTLA-4 blockade) compared to controls, suggesting that FSTL3 may be involved in immunotherapy resistance.In conclusion, this study suggests a role for FSTL3 as a prognostic marker and as therapeutic target in HGSOC, where it may play a role in promoting a mesenchymal tumor phenotype, maintaining an immunosuppressive tumor microenvironment, and driving immunotherapy resistance.HighlightsHigh FSTL3 levels are associated with poor outcomes in ovarian cancer.Serum levels of FSTL3 increase during tumor growth and reflect tumor burden and therapy response.Overexpression of FSTL3 in cancer cells promotes a fibrotic tumor microenvironment and immunocyte exclusion.Overexpression of FSTL3 in tumors induces resistance to Chk1 and immune checkpoint inhibitor combination therapy.Graphical abstract

Enhancing chemotherapy for pancreatic cancer through efficient and sustained tumor microenvironment remodeling with a fibroblast-targeted nanosystem

Pancreatic Ductal Adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related deaths in the US, with a five-year survival rate of 12%. Barriers to treatment include the highly fibrotic tumor microenvironment (TME), the high interstitial pressure which acts to collapse the blood vessels, and the influx of immunosuppressive immune cells. Within this immune milieu, we find that the most abundant are tumor-associated macrophages (TAMs). These TAMs display a phenotype similar to anti-inflammatory (M2-like) macrophages. Despite TAMs exerting an immunosuppressive function within the TME, there is not a complete overlap with the characteristics associated with M2 macrophages. Interestingly, TAMs still display many markers associated with the pro-inflammatory (M1-like) macrophages. This dichotomy suggests there may be a level of plasticity within the macrophage compartment that can be exploited to lead to better tumor control. Previous studies have shown a connection between arginine metabolism and macrophage effector function along the M1/M2 axis. In macrophages, arginine can be broken down in two ways: via iNOS into nitric oxide, which aids in effector function, or via Arginase 1 (Arg1) into ornithine and later proline, which is an essential component of the extracellular matrix (ECM). This study seeks to elucidate how metabolic perturbations influence macrophage function and phenotype. Here we observed that changes in oxygen availability and glucose abundance shifted macrophage expression of iNOS and Arg1. In vivo we found that conditionally knocking out Arg1 in the myeloid compartment leads to lower fibrosis when inducing inflammation in the pancreas. Furthermore, macrophages polarized with PDAC cancer-conditioned media display a sharp increase in Arg1 expression as compared to unpolarized macrophages, as well as increased resistance to both glycolytic inhibitors and mitochondrial inhibitors. This preliminary data demonstrates an important link between macrophage metabolism and function in the context of pancreatic disease. Citation Format: Cecily Anaraki, Christopher Halbrook. Metabolism as a key regulator of macrophage phenotype in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1599.

We seek to combine a unique innate immunomodulatory nanoparticle (NP) system with RAS inhibitors for a multi-faceted therapy that mitigates the formidable drug delivery challenges in pancreatic ductal adenocarcinoma (PDAC). PDAC has quickly risen to the third most deadly cancer largely due to its fibrotic, desmoplastic, and immunosuppressive tumor microenvironment (TME) that limits delivery and efficacy of chemo- and immunotherapeutics. We have engineered unique lipid-based NPs that co-encapsulate agonists of the Stimulator of Interferon Genes (STING) and Toll-like Receptor 4 (TLR4) pathways. NPs can be safely delivered in the systemic blood circulation to achieve TME deposition, uptake by innate antigen-presenting cells (APCs), and robust, synergistic production of Type I interferons by dual STING/TLR4 activation. Here, we hypothesize that combination therapy of proinflammatory NPs with tumor senescence-inducing RAS inhibitors, tremetinib and palbociclib, will not only augment CD8+ T cell recruitment to the TME but enhance their sustained activation by mitigating local immunosuppression. In mice bearing orthotopic transplant KPC tumors or KPC GEMMs, combination NP/inhibitor treatment promoted significant NP delivery to tumors, APC and natural killer (NK) cell activation, and CD8+ T cell-mediated clearance, compared to monotherapies and untreated controls. Unexpectedly and strikingly, these studies also showed that, besides innate immune cells, combination therapy also promoted Type I interferon and other proinflammatory cytokine production by PDAC tumor cells, which are otherwise notoriously unresponsive to current state-of-the-art treatments. Further, in KPC GEMMs, 25% of mice exhibited apparent complete responses following combination treatment, which, to our knowledge, is challenging to achieve in this model. Current studies include the mechanistic elucidation of immune- and tumor-intrinsic factors that govern efficacy of our combination therapy. In conclusion, these findings strongly corroborate the use of this NP-based system as a platform therapy for similar drugs and across other aggressive cancers. They also strongly make the case for the rational design of combination therapies to achieve synergistic therapeutic outcomes with minimal systemic toxicities. Citation Format: Prabhani Atukorale, Marcus Ruscetti, Loretah Chibaya, Christina Lusi, Kelly DeMarco, Griffin Kane, Meghan Brassil, Chaitanya Parikh, Katherine Murphy. Nanoparticle-mediated combination therapy to synergistically harness Type I interferons and senescence in the pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4043.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognostics and substantial therapeutic challenges, with dismal survival rates. Tumor resistance in PDAC is primarily attributed to its fibrotic, hypoxic, and immune-suppressive tumor microenvironment (TME). Endoscopic ultrasound-guided radiofrequency ablation (EUS-RFA), an Food and Drug Administration (FDA)-approved minimally invasive technique for treating pancreatic cancer, disrupts tumors with heat and induces coagulative necrosis, releasing tumor antigens that may trigger a systemic immune response—the abscopal effect. We aim to elucidate the roles of EUS-RFA-mediated thermal and mechanical stress in enhancing anti-tumor immunity in PDAC. A comprehensive literature review focused on radiofrequency immunomodulation and immunotherapy in pancreatic tumors to understand the pathophysiological mechanisms of RFA and its effect on the TME, which could prevent recurrence and resistance. We reviewed clinical, preclinical, and in vitro studies on RFA mechanisms in pancreatic adenocarcinoma, discussing the unique immunomodulatory effects of EUS-RFA. Recent findings suggest that combining RFA with immune adjuvants enhances responses in pancreatic adenocarcinoma. EUS-RFA offers a dual benefit against PDAC by directly reducing tumor viability and indirectly enhancing anti-tumor immunity. Observations of neutrophil-mediated immunomodulation and programmed cell death ligand 1 (PD-L1) modulation support integrating EUS-RFA with targeted immunotherapies for managing pancreatic adenocarcinoma. Integrating EUS-RFA in PDAC treatment promises direct cytoreduction and synergistic effects with molecular targeted therapies. Prospective clinical trials are crucial to assess the efficacy of this combined approach in improving outcomes and survival rates in advanced PDAC cases.