Oakley C. Olson

CSF-1R inhibition alters macrophage polarization and blocks glioma progression

Glioblastoma multiforme (GBM) comprises several molecular subtypes, including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumor-associated macrophages and microglia (TAMs). Macrophages depend on colony stimulating factor-1 (CSF-1) for differentiation and survival. We used an inhibitor of the CSF-1 receptor (CSF-1R) to target TAMs in a mouse proneural GBM model, which significantly increased survival and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, TAMs were not depleted in treated mice. Instead, glioma-secreted factors, including granulocyte-macrophage CSF (GM-CSF) and interferon-γ (IFN-γ), facilitated TAM survival in the context of CSF-1R inhibition. Expression of alternatively activated M2 markers decreased in surviving TAMs, which is consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in patients with proneural GBM. Our results identify TAMs as a promising therapeutic target for proneural gliomas and establish the translational potential of CSF-1R inhibition for GBM.

Macrophages and cathepsin proteases blunt chemotherapeutic response in breast cancer

The microenvironment is known to critically modulate tumor progression, yet its role in regulating treatment response is poorly understood. Here we found increased macrophage infiltration and cathepsin protease levels in mammary tumors following paclitaxel (Taxol) chemotherapy. Cathepsin-expressing macrophages protected against Taxol-induced tumor cell death in coculture, an effect fully reversed by cathepsin inhibition and mediated partially by cathepsins B and S. Macrophages were also found to protect against tumor cell death induced by additional chemotherapeutics, specifically etoposide and doxorubicin. Combining Taxol with cathepsin inhibition in vivo significantly enhanced efficacy against primary and metastatic tumors, supporting the therapeutic relevance of this effect. Additionally incorporating continuous low-dose cyclophosphamide dramatically impaired tumor growth and metastasis and improved survival. This study highlights the importance of integrated targeting of the tumor and its microenvironment and implicates macrophages and cathepsins in blunting chemotherapeutic response.

Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response

Cysteine cathepsin protease activity is frequently dysregulated in the context of neoplastic transformation. Increased activity and aberrant localization of proteases within the tumour microenvironment have a potent role in driving cancer progression, proliferation, invasion and metastasis. Recent studies have also uncovered functions for cathepsins in the suppression of the response to therapeutic intervention in various malignancies. However, cathepsins can be either tumour promoting or tumour suppressive depending on the context, which emphasizes the importance of rigorous in vivo analyses to ascertain function. Here, we review the basic research and clinical findings that underlie the roles of cathepsins in cancer, and provide a roadmap for the rational integration of cathepsin-targeting agents into clinical treatment.

Perivascular M2 Macrophages Stimulate Tumor Relapse after Chemotherapy

Tumor relapse after chemotherapy-induced regression is a major clinical problem, because it often involves inoperable metastatic disease. Tumor-associated macrophages (TAM) are known to limit the cytotoxic effects of chemotherapy in preclinical models of cancer. Here, we report that an alternatively activated (M2) subpopulation of TAMs (MRC1(+)TIE2(Hi)CXCR4(Hi)) accumulate around blood vessels in tumors after chemotherapy, where they promote tumor revascularization and relapse, in part, via VEGF-A release. A similar perivascular, M2-related TAM subset was present in human breast carcinomas and bone metastases after chemotherapy. Although a small proportion of M2 TAMs were also present in hypoxic tumor areas, when we genetically ablated their ability to respond to hypoxia via hypoxia-inducible factors 1 and 2, tumor relapse was unaffected. TAMs were the predominant cells expressing immunoreactive CXCR4 in chemotherapy-treated mouse tumors, with the highest levels expressed by MRC1(+) TAMs clustering around the tumor vasculature. Furthermore, the primary CXCR4 ligand, CXCL12, was upregulated in these perivascular sites after chemotherapy, where it was selectively chemotactic for MRC1(+) TAMs. Interestingly, HMOX-1, a marker of oxidative stress, was also upregulated in perivascular areas after chemotherapy. This enzyme generates carbon monoxide from the breakdown of heme, a gas known to upregulate CXCL12. Finally, pharmacologic blockade of CXCR4 selectively reduced M2-related TAMs after chemotherapy, especially those in direct contact with blood vessels, thereby reducing tumor revascularization and regrowth. Our studies rationalize a strategy to leverage chemotherapeutic efficacy by selectively targeting this perivascular, relapse-promoting M2-related TAM cell population.

Microenvironment-mediated resistance to anticancer therapies

Resistance to molecularly targeted therapies can result from genomic alterations in the tumor cells that reactivate oncogenic signaling. Less is known of tumor cell-extrinsic mechanisms of resistance to targeted therapies. Two recent studies have identified HGF as a soluble factor capable of mediating resistance to BRAF and HER2 inhibitors in a paracrine manner. These new findings suggest an important role for the tumor microenvironment in mediating resistance to molecularly targeted therapies.

Obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through IL5 and GM-CSF

Obesity is associated with chronic, low-grade inflammation, which can disrupt homeostasis within tissue microenvironments. Given the correlation between obesity and relative risk of death from cancer, we investigated whether obesity-associated inflammation promotes metastatic progression. We demonstrate that obesity causes lung neutrophilia in otherwise normal mice, which is further exacerbated by the presence of a primary tumour. The increase in lung neutrophils translates to increased breast cancer metastasis to this site, in a GM-CSF- and IL5-dependent manner. Importantly, weight loss is sufficient to reverse this effect, and reduce serum levels of GM-CSF and IL5 in both mouse models and humans. Our data indicate that special consideration of the obese patient population is critical for effective management of cancer progression.

Tumor-Associated Macrophages Suppress the Cytotoxic Activity of Antimitotic Agents

SUMMARY Antimitotic agents, including Taxol, disrupt microtubule dynamics and cause a protracted mitotic arrest and subsequent cell death. Despite the broad utility of these drugs in breast cancer and other tumor types, clinical response remains variable. Tumor-associated macrophages (TAMs) suppress the duration of Taxol-induced mitotic arrest in breast cancer cells and promote earlier mitotic slippage. This correlates with a decrease in the phosphorylated form of histone H2AX (γH2AX), decreased p53 activation, and reduced cancer cell death in interphase. TAMs promote cancer cell viability following mitotic slippage in a manner sensitive to MAPK/ERK kinase (MEK) inhibition. Acute depletion of major histocompatibility complex class II low (MHCIIlo) TAMs increased Taxol-induced DNA damage and apoptosis in cancer cells, leading to greater efficacy in intervention trials. MEK inhibition blocked the protective capacity of TAMs and phenocopied the effects of TAM depletion on Taxol treatment. TAMs suppress the cytotoxic effects of Taxol, in part through cell non-autonomous modulation of mitotic arrest in cancer cells, and targeting TAM-cancer cell interactions potentiates Taxol efficacy.

Inflammatory monocytes promote perineural invasion via CCL2-mediated recruitment and cathepsin B expression

Perineural invasion (PNI) is an ominous event strongly linked to poor clinical outcome. Cells residing within peripheral nerves collaborate with cancer cells to enable PNI, but the contributing conditions within the tumor microenvironment are not well understood. Here, we show that CCR2-expressing inflammatory monocytes (IM) are preferentially recruited to sites of PNI, where they differentiate into macrophages and potentiate nerve invasion through a cathepsin B-mediated process. A series of adoptive transfer experiments with genetically engineered donors and recipients demonstrated that IM recruitment to nerves was driven by CCL2 released from Schwann cells at the site of PNI, but not CCL7, an alternate ligand for CCR2. Interruption of either CCL2-CCR2 signaling or cathepsin B function significantly impaired PNI in vivo Correlative studies in human specimens demonstrated that cathepsin B-producing macrophages were enriched in invaded nerves, which was associated with increased local tumor recurrence. These findings deepen our understanding of PNI pathogenesis and illuminate how PNI is driven in part by corruption of a nerve repair program. Further, they support the exploration of inhibiting IM recruitment and function as a targeted therapy for PNI. Cancer Res; 77(22); 6400-14. ©2017 AACR.

A Splicing Twist on Metastasis

An oncogenic splice variant of the transcription factor KLF6 is associated with metastasis and poor survival in node-negative breast cancer. Abstract An oncogenic splice variant of the transcription factor KLF6 is associated with metastasis and poor survival in node-negative breast cancer patients and promotes the epithelial-to-mesenchymal transition by regulating Twist.

Abstract 549: Sensitization to chemotherapy by inhibition of cathepsin proteases in a mouse model of metastatic breast cancer

A growing body of evidence supports the role of the tumor microenvironment in promoting malignant progression and modulating the response to anti-cancer therapy. Tumor associated macrophages (TAMs) have been established as an important component of the tumor microenvironment, contributing to angiogenesis, matrix remodeling, invasion and metastasis. We and others have identified cysteine cathepsin proteases as key effectors of these pro-tumorigenic functions of TAMs. These same processes, which are integral to tumor progression, may also aid tumors in recovering from cytotoxic insults, thereby blunting our therapeutic efforts. There is thus a strong rationale for combinatorial targeting of both the tumor and the supporting stroma. We have uncovered such a phenomenon in a mouse model of metastatic mammary carcinogenesis (MMTV-PyMT). Following maximum tolerated dose paclitaxel treatment (TaxMTD), we observed an elevation in levels of circulating cathepsin-activity+ myeloid cells and a subsequent increase in macrophages and cathepsin activity levels in the tumor. In co-cultures, cathepsin-activity+ macrophages reduced paclitaxel-induced tumor cell death. Furthermore, the use of primary macrophages from various cathepsin null genetic backgrounds identified cathepsins B and S as mediators of this effect. To determine whether cathepsin inhibition could therefore enhance the effects of chemotherapy, we treated MMTV-PyMT mice with both TaxMTD and a pan-cathepsin inhibitor, JPM. While JPM alone had no effect on mammary tumor burden, it significantly impaired tumor growth when combined with TaxMTD, demonstrating that the TaxMTD-induced elevation in tumor cathepsin activity is functionally relevant. To further our goal of simultaneous targeting of tumor and stroma, we added a low-dose cyclophosphamide regimen, which has been shown to have both anti-angiogenic and immune-stimulatory properties. This triple combination treatment was substantially more effective than any double or single drug combination. Importantly, metastatic burden was also significantly reduced in triple-treated mice as compared to controls, and long-term survival was improved. These studies suggest cathepsin+ macrophages are recruited to the tumor after TaxMTD treatment to promote tumor survival and recovery. The efficacy of combining cathepsin inhibition with paclitaxel highlights both the importance of integrated therapeutic targeting of tumor and stroma, as well as the role of TAMs in modulating resistance to chemotherapy. The addition of cysteine cathepsin inhibition to chemotherapeutic regimens thus holds considerable promise for clinical translation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 549. doi:10.1158/1538-7445.AM2011-549