Michelle Dotzert, PhD
The tumor microenvironment consists of all cellular and non-cellular components surrounding the tumor. It includes an extracellular matrix (ECM), fibroblasts, mesenchymal stromal cells, pericytes, adipocytes, vasculature, lymphatic networks, and immune cells. The communication between tumor cells and the microenvironment leads to proliferation and metastasis.
Examining the tumor microenvironment and understanding its evolution during tumor development is enabling researchers to identify potential therapeutic targets and biomarkers for liquid biopsy. The following are therapeutic targets that have been identified and corresponding treatment strategies.
The Extracellular Matrix
The extracellular matrix (ECM) consists of collagen, elastin, fibronectin, hyaluronic acid, proteoglycans, and glycoproteins, and it contains numerous growth factors. The ECM is crucial for cancer spread and metastasis, as adhering cells are moved into or out of the tumor microenvironment.
Targeting the ECM may enable chemotherapy drugs to access the tumor, increasing their effectiveness. Transforming growth factor beta (TGF-β1) upregulates expression of matrix proteins within the ECM, resulting in desmoplasia. Inhibiting the TGF-β pathway with FDA approved angiotensin II receptor agonists, such as Losartan, may alleviate pressure within the tumor microenvironment, restoring blood flow for delivery of chemotherapeutic drugs. Similarly, SST0001 (Roneparstat) is a heparinase that participates in ECM degradation and remodeling and may be effective against the ECM.
Matrix metalloproteinases (MMPs) deplete the matrix, and contribute to cancer stem cell (CSC) formation. Incyclinide (COL-3) is an MMP inhibitor that has undergone clinical trials for the treatment of advanced solid tumors and recurrent high-grade astrocytoma.
ECM protein deposition results in fibrosis, potential vascular occlusion, and subsequent hypoxia. Hypoxia induces a cellular response within the tumor microenvironment orchestrated by hypoxia-induced factor-1 (HIF-1). HIF-1 signaling enables cancer cell adaptation and metastasis via alterations in glucose metabolism, angiogenesis, and macrophage polarization.
Topotecan received FDA approval for the treatment of ovarian and small cell lung cancer. It is a HIF-1α protein translation inhibitor, that poisons topoisomerase I via induction of stable Top1-DNA cleavage complexes. In the presence of DNA replication, it generates double strand DNA breaks and cytotoxicity. Temsirolimus and Everolimus are approved for treatment of renal cancer, as they prevent HIF-1α translation via mTOR inhibition.
Other HIF-1α targeted treatments undergoing trials include Digoxin, a HIF-1α translation inhibitor being evaluated for the treatment of head and neck, breast, prostate, and non-small cell lung cancer. The tissue oxygenation effects of Metformin, a first-line treatment for type 2 diabetes, are being tested for head and neck squamous cell carcinoma.
The Warburg effect is the observation that even in aerobic conditions, tumors favor ATP production via anaerobic glycolysis over oxidative phosphorylation. This shift is thought to be mediated by HIF-1α transcriptional regulation. Anaerobic ATP production results in accumulation of lactic and carbonic acids. Tumor cells express H+-ATPases, Na+/H+ exchangers, monocarboxylate transporters, carbonic anhydrase IX, and Na+/HCO3 co-transporters to prevent proton accumulation and toxic acidification. Increased acidity in the tumor microenvironment impairs immune cell resistance to tumor progression, and prevents chemotherapeutic agents from entering the tumor microenvironment.
Treatments target proton exchangers and transporters in the tumor microenvironment. Acetazolamide is a carbonic anhydrase inhibitor, currently being studied in combination with the chemotherapeutic drug Temozolomide for malignant glioma of the brain. It is also being studied in combination with radiochemotherapy for the treatment of small cell lung cancer. Another carbonic anhydrase inhibitor, SLC-0111 has been studied for the treatment of advanced solid tumors, and trials (not yet recruiting) will also examine it in combination with Gemcitabine for metastatic pancreatic ductal adenocarcinoma.
Tumor-associated macrophages (TAMs) are converted from macrophages originating from the embryonic yolk sac laid down in tissues during development, or from monocytes attracted by signaling molecules including colony-stimulating factor 1 (CSF-1) and CC chemokine ligand 2 (CCL2). TAMs secrete cytokines into the tumor microenvironment to support tumor growth and proliferation via inflammation, immune regulation, angiogenesis, and metastasis.
Myeloid-derived suppressor cells (MDSC) are also a component of the tumor microenvironment. MDSC production in bone marrow is stimulated by cancer cell vascular endothelial growth factor (VEGF) production. These cells then migrate to the tumor microenvironment where they promote proliferation, and vascularization, and exhibit immune suppressive activity.
Ligand binding to colony-stimulating factor 1 receptor positive (CSF1R+) macrophages induces receptor signaling and differentiation. Pexidartinib is a CSF1R inhibitor, FDA approved for the treatment of symptomatic tenosynovial giant cell tumor (TGCT). Several other trials examine the effects of CSF1R inhibitors in cancer, including ARRY-382, BLZ945, JNJ-40346527, and DCC-3014.
CSF1R antibodies are also being examined. Emactuzumab is a humanized monoclonal antibody directed against CSF1R being investigated for use in patients with metastatic pancreatic ductal adenocarcinoma (PDAC). Others include Cabiralizumab, SNDX-6352, PD-0360324, and Lacnotuzumab.
Chiauranib is a small molecule inhibitor of serine-threonine kinases including VEGFRs, and Nilotinib inhibits receptor tyrosine kinaes including CSF1R.
Inflammatory cytokines secreted by TAMs are another potential target for treatment. Anakinra is an interleukin-1 (IL-1) receptor agonist. It’s binding to the IL-1 receptor has been shown to inhibit VEGF and other pro-angiogenic factors. Similarly, the monoclonal antibody Canakinumab targets IL-1β, preventing it from binding the IL-1 receptor and suppressing the inflammatory response.
Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells in the tumor microenvironment. They result from fibroblast differentiation in response to growth factors. CAFs promote angiogenesis via VEGF, CXCL12a and FGF-2 production, and modulate the immune response via macrophage infiltration and cell polarization.
Fibroblast activation protein alpha (FAP) is highly expressed on stromal fibroblasts but not healthy tissues. RO6874281 is a recombinant fusion protein consisting of a human monoclonal antibody directed against FAP and a variant interleukin-2. Upon binding to FAP, the IL-2 moiety activates natural killer and cytotoxic T-cells. RO6874281 is currently being assessed in several recruiting and active trials for advanced or metastatic solid tumors, breast cancer, cancer of the head and neck, pancreatic adenocarcinoma, metastatic melanoma, and renal cell carcinoma. Another trial is investigating the safety of a single dose of adoptively transferred FAP-specific CD8 positive re-directed T cells in patients with malignant pleural mesothelioma.
Exosomes are extracellular vesicles ranging from 30-150 nm, that contain DNA, RNA, proteins, and membrane bound factors. Tumor cell-derived exosomes modulate the tumor microenvironment via paracrine signaling. They also modulate recipient cell phenotype and stimulate healthy cells to undergo a malignant transition. Exosome miRNA transport contributes to angiogenesis, CAF differentiation, inflammation, and modulation of the immune response to promote tumor growth and metastasis.
Mesenchymal stromal cells-derived exosomes with KrasG12D siRNA (iExosomes) are being studied for the treatment pancreatic cancer with KrasG12D mutation. Mutant GTPase KRAS drives the development of pancreatic cancer, and iExosomes that target oncogenic KRAS may be an effective treatment strategy.
Exosomes are also being studied as potential candidates for liquid biopsy. The performance of the ExoDx Prostate (IntelliScore), a urine exosome gene expression assay, is being assessed to determine its potential to reduce the number of initial prostate biopsies for men with elevated Prostate-Specific Antigen (PSA). As part of a trial evaluating the efficacy and safety of combined Ipilimumab and Nivolumab for breast cancer, the predictive value of circulating cell-free tumor DNA (ctDNA) and immune signature by exosome analysis via blood sample will be examined.
Like organs, tumors require and adequate blood supply. Several growth factors in the tumor microenvironment promote angiogenesis, including vascular endothelial growth factors (VEGFs). However, intratumoral vessels are irregular with a leaky lumen that compromises flow, resulting in hypoxia that potentiates tumor development. Drug delivery is also impaired with inefficient blood flow. In addition to pro-angiogenic effects, VEGF is also immunosuppressive, exerting effects on effector T cells and dendritic cells.
Preventing neovascularization is achieved by targeting VEGF, VEGF receptors (VEGFR), and other vascularization pathways. Several VEGF and VEGFR inhibitors are approved to treat various cancers, and many other trials are underway.
Cediranib binds and inhibits VEGFR-1, -2, and -3, and is being assessed in active clinical trials for ovarian, fallopian tube, and primary peritoneal cancers, as well as advanced solid tumors, and metastatic soft tissue sarcoma. Ramucirumab is a recombinant human monoclonal antibody that inhibits VEGFR-2, and is approved for colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, and stomach adenocarcinoma. Pazopanib, a VEGFR-1, -2, and -3 inhibitor is approved for renal cell carcinoma and soft tissue sarcoma.
Bevacizumab is a recombinant humanized monoclonal antibody against VEGF, approved for cervical, colorectal, nonsquamous non-small cell lung cancer, ovarian epithelial, fallopian tube or primary peritoneal cancer, renal cell carcinoma, and glioblastoma. Aflibercept also inhibits VEGF, although it functions as a soluble decoy receptor to prevent VEGFs from binding VEGFRs. It consists of the extracellular domains of human VEGFR1 and VEGFR2 fused to the constant region of human IgG1, and is approved to treat colorectal cancer.
Everolimus is also used to target neovascularization, however unlike VEGF and VEGFR inhibitors, it binds to the immunophilin FK Binding Protein-12 (FKBP-12), creating an immunosuppressive complex that binds and inhibits mammalian Target of Rapamycin (mTOR). mTOR inhibition reduces endothelial cell proliferation via the mTOR/AP-1/VEGF pathway. Everolimus is approved to treat breast, pancreatic, gastrointestinal, and lung cancer, renal cell carcinoma, and subpendymal giant cell astrocytoma.