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RNA labeling in situ has enormous potential to visualize transcripts and quantify their levels in single cells, but it remains challenging to produce high levels of signal while also enabling multiplexed detection of multiple RNA species simultaneously. Here, we describe clampFISH 2.0, a method that uses an inverted padlock design to efficiently detect and exponentially amplify signals from many RNA species at once, while also reducing time and cost compared to the prior clampFISH method. We leverage the increased throughput afforded by multiplexed signal amplification and sequential detection by demonstrating the ability to detect 10 different RNA species in over 1 million cells. We also show that clampFISH 2.0 works in tissue sections. We expect the advantages offered by clampFISH 2.0 will enable many applications in spatial transcriptomics.

Aged melanoma patients (>65 years old) have more aggressive disease relative to young patients (5-fold levels of insulin-like growth factor binding protein 2 (IGFBP2) in the aged fibroblast secretome. IGFBP2 functionally triggers upregulation of the PI3K-dependent fatty acid biosynthesis program in melanoma cells through increases in FASN. Melanoma cells co-cultured with aged dermal fibroblasts have higher levels of lipids relative to young dermal fibroblasts, which can be lowered by silencing IGFBP2 expression in fibroblasts, prior to treating with conditioned media. Conversely, ectopically treating melanoma cells with recombinant IGFBP2 in the presence of conditioned media from young fibroblasts, promoted lipid synthesis and accumulation in the melanoma cells. Neutralizing IGFBP2in vitroreduces migration and invasion in melanoma cells, and invivostudies demonstrate that neutralizing IGFBP2 in syngeneic aged mice, ablates tumor growth as well as metastasis. Conversely, ectopic treatment of young mice with IGFBP2 in young mice increases tumor growth and metastasis. Our data reveal that aged dermal fibroblasts increase melanoma cell aggressiveness through increased secretion of IGFBP2, stressing the importance of considering age when designing studies and treatment.SignificanceThe aged microenvironment drives metastasis in melanoma cells. This study reports that IGFBP2 secretion by aged fibroblasts induces FASN in melanoma cells and drives metastasis. Neutralizing IGFBP2 decreases melanoma tumor growth and metastasis.

Supplementary Figures and Legends.

Young BODIPY-C12 fibroblasts with GFP-Tagged melanoma cells showing lipid uptake

Aged BODIPY-C12 fibroblasts with GFP-tagged melanoma cells

Supplementary Figures S1-S4

Chemical Methods

Supplementary Figure 2

Figure S2 shows astrocytes facilitate the growth and survival of brain metastatic cancer cells.

Older patients with melanoma (>50 years old) have poorer prognoses and response rates to targeted therapy compared with young patients (Significance:These data show that melanoma cells take up lipids from aged fibroblasts, via FATP2, and use them to resist targeted therapy. The response to targeted therapy is altered in aged individuals because of the influences of the aged microenvironment, and these data suggest FATP2 as a target to overcome resistance.See related commentary by Montal and White, p. 1255..This article is highlighted in the In This Issue feature, p. 1241

Supplemental Chemical Methods

Brain metastasis, the most lethal form of melanoma and carcinoma, is the consequence of favorable interactions between the invading cancer cells and the brain cells. Peroxisome proliferator–activated receptor γ (PPARγ) has ambiguous functions in cancer development, and its relevance in advanced brain metastasis remains unclear. Here, we demonstrate that astrocytes, the unique brain glial cells, activate PPARγ in brain metastatic cancer cells. PPARγ activation enhances cell proliferation and metastatic outgrowth in the brain. Mechanistically, astrocytes have a high content of polyunsaturated fatty acids that act as “donors” of PPARγ activators to the invading cancer cells. In clinical samples, PPARγ signaling is significantly higher in brain metastatic lesions. Notably, systemic administration of PPARγ antagonists significantly reduces brain metastatic burden in vivo. Our study clarifies a prometastatic role for PPARγ signaling in cancer metastasis in the lipid-rich brain microenvironment and argues for the use of PPARγ blockade to treat brain metastasis.Significance:Brain-tropic cancer cells take advantage of the lipid-rich brain microenvironment to facilitate their proliferation by activating PPARγ signaling. This protumor effect of PPARγ in advanced brain metastases is in contrast to its antitumor function in carcinogenesis and early metastatic steps, indicating that PPARγ has diverse functions at different stages of cancer development.This article is highlighted in the In This Issue feature, p. 1631

Aged BODIPY-C12 fibroblasts with GFP-tagged melanoma cells

Supplemental Figures and Table

Young BODIPY-C12 fibroblasts with GFP-Tagged melanoma cells showing lipid uptake

Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an in situ photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives.Significance:This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer.This article is highlighted in the In This Issue feature, p. 151

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Supplementary Table

Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell–like pathways hijacked by tumor cells.Significance:This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance.

Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells—in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state(1–3). It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing(4–8). We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.

Supplementary Tables

Supplementary Figure 4

Supplementary Figure 7 - Pro- and Anti-Tumor Effects of IFN-g Signalling Within The TME

Purpose: Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma.Experimental Design: PPARγ increases klotho levels and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/Wnt5A cross-talk, in vitro and in vivo, and the implications of that for targeted therapy in young versus aged animals.Results: We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF inhibitor–sensitive and BRAF inhibitor–resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors.Conclusions: Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types. Clin Cancer Res; 23(12); 3181–90. ©2017 AACR.

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Supplementary Figure 5

Supplementary Figure 2

PDF containing all supplementary figures. Supplementary Figure Legends. Supplemental Figure 1. A, graph indicating the number of patients for which best response data to pembrolizumab was obtained, and from which institution. B, various response groups of patients treated with pembrolizumab, separated by gender, and using 62 years of age as the most statistically significant cut-off. Supplemental Figure 2. A, B Change in mouse weight on final day of experiment from start in female and male mice bearing BSC9AJ2 tumors, after treatment with 3 doses of 300µg rat IgG2AK (N=5) or 4 doses of 300µg anti-PD1 (N=5) every 5 days, starting on day 0. Supplemental Figure 3. A, CD45+ cells within Yumm1.7 and B. BSC9AJ2 tumors. Err=95%CI. B, CD45+ cells within in tumors of Yumm1.7 and Yumm2.1 tumors. C, CD8b+ cells following 5 hour incubation with PMA and ionomycin analyzed by FACS analysis. Err=SD. D, E CD45+CD8b+ cells expressing TNFα and IFNγ within spleens from mice harboring Yumm1.7 or BSC9AJ2 tumors. Err=95%CI. Significance was determined using individual two-tailed student's t-test assuming unequal variance for 1-2 factors and using a 2-way ANOVA for 3 or more factors. Supplemental Figure 4. A 10X view with 40X zoom of FOXP3+ foci staining in melanoma patient biopsies. B, FOXP3+ staining from patient tumors across various age groups. C, CD8+ staining from patient tumors across various age groups. D, percentages of patients in the indicated age groups, whose CD8:FOXP3 ratio is low (< median - 1), med (median +/- 1), or high (> median + 1). Supplemental Figure 5. A. Mouse weight on final day of experiment from start in mice treated with anti-CD25 or an IgG1A isotype 5 days prior to sub-dermal of BSC9AJ2 cells with treatments continuing every 5 days until sacrifice. Anti-PD1 was administered every 3-4 days, starting on day 9 post-tumoral injection. Err=95%CI. Statistical significance determined by 2 way ANOVA. Err bars = SEM. B. Tumor growth for individual mice, per condition in A. Supplemental Figure 6. A. Ratios of CD3+CD8a+ to CD3+CD4+FoxP3+ in BSC9AJ2 tumors from young female mice 4 days following cyclophosphamide intraperitoneal injection at indicated doses. 5 mice in PBS group, and 3 mice per dose. Err=SEM. B, intra-tumoral CD3+CD4+FoxP3+ populations. Err=SD. C, tumor growth in young female mice bearing BSC9AJ2 tumors were injected 6 days post tumor implantation with either 100µL PBS control, or 100µL PBS containing 25 mgs/kg cyclophosphamide intra-peritoneally. Four days later, mice were also given either 300µg anti-PD1 or vehicle control. A total of 4 doses of PD1 was given on day 0, 5, 10, and 14. Statistical significance was determined using a linear mixed-effect model. Err.=SEM. D, fold change in mouse weight from C. Err=SD. E. Intra-tumoral ratios of CD3+CD8a+ to CD3+CD4+FoxP3+ at the end of the experiment. Err.=SEM.

upplementary Figure 1 - Wnt5A Decreases Proliferation Specifically in Tumors Via the Recruitment of MDSCs

Supplementary Figure 1

Figure Legends for Supplemental Data

Supplementary Figure 4 - rWnt5A and Myeloid-Wnt5A Promotes an Immunosuppressive TME in Peripheral Blood and Lungs in vivo

Supplemental Table 1. Antibodies used.

Purpose:Angiogenesis is thought to be critical for tumor metastasis. However, inhibiting angiogenesis using antibodies such as bevacizumab (Avastin), has had little impact on melanoma patient survival. We have demonstrated that both angiogenesis and metastasis are increased in older individuals, and therefore sought to investigate whether there was an age-related difference in response to bevacizumab, and if so, what the underlying mechanism could be.Experimental Design:We analyzed data from the AVAST-M trial of 1,343 patients with melanoma treated with bevacizumab to determine whether there is an age-dependent response to bevacizumab. We also examined the age-dependent expression of VEGF and its cognate receptors in patients with melanoma, while using syngeneic melanoma animal models to target VEGF in young versus old mice. We also examined the age-related proangiogenic factor secreted frizzled-related protein 2 (sFRP2) and whether it could modulate response to anti-VEGF therapy.Results:We show that older patients respond poorly to bevacizumab, whereas younger patients show improvement in both disease-free survival and overall survival. We find that targeting VEGF does not ablate angiogenesis in an aged mouse model, while sFRP2 promotes angiogenesis in vitro and in young mice. Targeting sFRP2 in aged mice successfully ablates angiogenesis, while the effects of targeting VEGF in young mice can be overcome by increasing sFRP2.Conclusions:VEGF is decreased during aging, thereby reducing response to bevacizumab. Despite the decrease in VEGF, angiogenesis is increased because of an increase in sFRP2 in the aged tumor microenvironment. These results stress the importance of considering age as a factor for designing targeted therapies.

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1.Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies.Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice.Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347–56. ©2018 AACR.See related commentary by Pawelec, p. 5193

Supplementary Figure 6 - rWnt5A Does Not Promote Treg Migration in vitro

Supplementary Figure 5 - Myeloid Wnt5A Does Not Influence CXCR2 Expression on MDSCs

Supplementary Figure 3 - Effects of Myeloid Wnt5A Knockdown on Apoptosis, and Wnt5A Expression in Cell lines and Tumor Lysates

Supplementary Figure 3

Supplementary Data from sFRP2 Supersedes VEGF as an Age-related Driver of Angiogenesis in Melanoma, Affecting Response to Anti-VEGF Therapy in Older Patients

Resistance to combination BRAF/MEK inhibitor (BRAFi/MEKi) therapy arises in nearly every patient withBRAFV600E/Kmelanoma, despite promising initial responses. Achieving cures in this expanding BRAFi/MEKi-resistant cohort represents one of the greatest challenges to the field; few experience additional durable benefit from immunotherapy and no alternative therapies exist. To better personalize therapy in cancer patients to address therapy relapse, umbrella trials have been initiated whereby genomic sequencing of a panel of potentially actionable targets guide therapy selection for patients; however, the superior efficacy of such approaches remains to be seen. We here test the robustness of the umbrella trial rationale by analyzing relationships between genomic status of a gene and the downstream consequences at the protein level of related pathway, which find poor relationships between mutations, copy number amplification, and protein level. To profile candidate therapeutic strategies that may offer clinical benefit in the context of acquired BRAFi/MEKi resistance, we established a repository ofpatient-derivedxenograft models from heavily pretreated patients with resistance to BRAFi/MEKi and/or immunotherapy (R-PDX). With these R-PDXs, we executedin vivocompound repurposing screens using 11 FDA-approved agents from an NCI-portfolio with pan-RTK, non-RTK and/or PI3K-mTOR specificity. We identify dasatinib as capable of restoring BRAFi/MEKi antitumor efficacy in ∼70% of R-PDX tested. A systems-biology analysis indicates elevated baseline protein expression of canonical drivers of therapy resistance (e.g., AXL, YAP, HSP70, phospho-AKT) as predictive of MAPKi/dasatinib sensitivity. We therefore propose that dasatinib-based MAPKi therapy may restore antitumor efficacy in patients that have relapsed to standard-of-care therapy by broadly targeting proteins critical in melanoma therapy escape. Further, we submit that this experimental PDX paradigm could potentially improve preclinical evaluation of therapeutic modalities and augment our ability to identify biomarker-defined patient subsets that may respond to a given clinical trial.SINGLE SENTENCE SUMMARYBroad target inhibition effective as a salvage strategy in BRAF/MEK inhibitor-acquired resistance PDX

Last October, within the 2021 SMR congress, we held the inaugural Diversity in Science Session. The goal of the session was to discuss diversity, equity, and inclusion in the melanoma research community and strategies to promote the advancement of underrepresented melanoma researchers. An international survey was conducted to assess the diversity, equity, and inclusion (DEI) climate among researchers and clinicians within the Society for Melanoma Research (SMR). The findings suggest there are feelings and experiences of inequity, bias, and harassment within the melanoma community that correlate with one’s gender, ethnic/racial group, and/or geographic location. Notably, significant reports of inequity in opportunity, discrimination, and sexual harassment demonstrate there is much work remaining to ensure all scientists in our community experience an academic workplace culture built on mutual respect, fair access, inclusion, and equitable opportunity.

Metastatic dissemination remains a significant barrier to successful therapy for melanoma. Wnt5A is a potent driver of invasion in melanoma and is believed to be secreted from the tumor microenvironment (TME). Our data suggest that myeloid-derived suppressor cells (MDSC) in the TME are a major source of Wnt5A and are reliant upon Wnt5A for multiple actions. Knockdown of Wnt5A specifically in the myeloid cells demonstrated a clear decrease in Wnt5A expression within the TME in vivo as well as a decrease in intratumoral MDSC and regulatory T cell (Treg). Wnt5A knockdown also decreased the immunosuppressive nature of MDSC and decreased expression of TGFβ1 and arginase 1. In the presence of Wnt5A-depleted MDSC, tumor-infiltrating lymphocytes expressed decreased PD-1 and LAG3, suggesting a less exhausted phenotype. Myeloid-specific Wnt5A knockdown also led to decreased lung metastasis. Tumor-infiltrating MDSC from control animals showed a strong positive correlation with Treg, which was completely ablated in animals with Wnt5A-negative MDSC. Overall, our data suggest that while MDSC contribute to an immunosuppressive and less immunogenic environment, they exhibit an additional function as the major source of Wnt5A in the TME. Significance: These findings demonstrate that myeloid cells provide a major source of Wnt5A to facilitate metastatic potential in melanoma cells and rely on Wnt5A for their immunosuppressive function.

Cellular plasticity describes the ability of cells to transition from one set of phenotypes to another. In melanoma, transient fluctuations in the molecular state of tumor cells mark the formation of rare cells primed to survive BRAF inhibition and reprogram into a stably drug-resistant fate. However, the biological processes governing cellular priming remain unknown. We used CRISPR-Cas9 genetic screens to identify genes that affect cell fate decisions by altering cellular plasticity. We found that many factors can independently affect cellular priming and fate decisions. We discovered a new plasticity-based mode of increasing resistance to BRAF inhibition that pushes cells towards a more differentiated state. Manipulating cellular plasticity through inhibition of DOT1L before the addition of the BRAF inhibitor resulted in more therapy resistance than concurrent administration. Our results indicate that modulating cellular plasticity can alter cell fate decisions and may prove useful for treating drug resistance in other cancers.

Plasticity enables cells to change their gene expression state in the absence of a genetic change. At the single-cell level, these gene expression states can persist for different lengths of time which is a quantitative measurement referred to as gene expression memory. Because plasticity is not encoded by genetic changes, these cell states can be reversible, and therefore, are amenable to modulation by disrupting gene expression memory. However, we currently do not have robust methods to find the regulators of memory or to track state switching in plastic cell populations. Here, we developed a lineage tracing-based technique to quantify gene expression memory and to identify single cells as they undergo cell state transitions. Applied to human melanoma cells, we quantified long-lived fluctuations in gene expression that underlie resistance to targeted therapy. Further, we identified the PI3K and TGF-β pathways as modulators of these state dynamics. Applying the gene expression signatures derived from this technique, we find that these expression states are generalizable to in vivo models and present in scRNA-seq from patient tumors. Leveraging the PI3K and TGF-β pathways as dials on memory between plastic states, we propose a “ pretreatment” model in which we first use a PI3K inhibitor to modulate the expression states of the cell population and then apply targeted therapy. This plasticity informed dosing scheme ultimately yields fewer resistant colonies than targeted therapy alone. Taken together, we describe a technique to find modulators of gene expression memory and then apply this knowledge to alter plastic cell states and their connected cell fates.

Purpose: Angiogenesis is thought to be critical for tumor metastasis. However, inhibiting angiogenesis using antibodies such as bevacizumab (Avastin), has had little impact on melanoma patient survival. We have demonstrated that both angiogenesis and metastasis are increased in older individuals, and therefore sought to investigate whether there was an age-related difference in response to bevacizumab, and if so, what the underlying mechanism could be. Experimental Design: We analyzed data from the AVAST-M trial of 1,343 patients with melanoma treated with bevacizumab to determine whether there is an age-dependent response to bevacizumab. We also examined the age-dependent expression of VEGF and its cognate receptors in patients with melanoma, while using syngeneic melanoma animal models to target VEGF in young versus old mice. We also examined the age-related proangiogenic factor secreted frizzled-related protein 2 (sFRP2) and whether it could modulate response to anti-VEGF therapy. Results: We show that older patients respond poorly to bevacizumab, whereas younger patients show improvement in both disease-free survival and overall survival. We find that targeting VEGF does not ablate angiogenesis in an aged mouse model, while sFRP2 promotes angiogenesis in vitro and in young mice. Targeting sFRP2 in aged mice successfully ablates angiogenesis, while the effects of targeting VEGF in young mice can be overcome by increasing sFRP2. Conclusions: VEGF is decreased during aging, thereby reducing response to bevacizumab. Despite the decrease in VEGF, angiogenesis is increased because of an increase in sFRP2 in the aged tumor microenvironment. These results stress the importance of considering age as a factor for designing targeted therapies.