The following articles have been recommended for further reading in the field of cancer immunotherapy by Dr. Pedro J. Romero, JITC Editor-in-Chief.
“Metabolic modulation of tumours with engineered bacteria for immunotherapy” by Fernando P Canale et al Nature 598, 662–666 (2021)
L-arginine is an amino acid essential to many biological processes, including immune cell proliferation and function. Fernando P Canale and colleagues investigated if increasing intratumoral L-arginine could work synergistically with immune checkpoint blockade to reduce tumor burden. In a mouse xenograft MC38 colon adenocarcinoma model, oral supplementation with L-arginine in combination with anti-PD-L1 therapy increased survival. The required dose of oral L-arginine supplementation was too high to be feasible for clinical use in humans, however. In order to continuously deliver L-arginine to the tumor microenvironment, the group developed an Escherichia coli Nissle 1917 (ECN) strain of bacteria, which was genetically engineered to be resistant to negative feedback for L-arginine synthesis or repression (L-Arg bacteria). With these alterations, the L-Arg bacteria colonized tumors after direct injection and converted ammonia (often produced in excess by tumor cells) into L-arginine within the tumor microenvironment. When combined with anti-PD-L1, more CD8+ and CD4+ T cells infiltrated the tumor compared to untreated or ECN-colonized tumors, and the infiltrating cells expressed higher levels of TNF and less PD-L1 and LAG3. The combination of PD-L1 blockade therapy and L-Arg colonization was also effective in reducing tumor growth and eradicating tumors in 74% of mice, a response rate that was roughly 30% higher than mice treated with anti-PD-L1 alone or with non-engineered ECN. Long-term anti-tumor immunity was also established in some mice. Importantly, intravenous L-Arg treatment was also effective at colonizing large tumors (> 100 mm3), and when combined with anti-PD-L1 therapy was able to reduce tumor growth.
“Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity” by Giusy Di Conza et al - Nature Immunology 2021 Oct 22. doi: 10.1038/s41590-021-01047-4
Tumor cells secrete factors that can influence the polarization of tumor-associated macrophages (TAMs) to be pro- or anti-tumorigenic in the tumor microenvironment, however, the pathways involved in this phenotypic plasticity have remained largely unknown. Giusy Di Conza and colleagues describe a mechanism related to endoplasmic reticulum (ER) stress and changes in lipid composition in pro-tumorigenic (M2) TAMs. Compared to splenic macrophages and anti-tumorigenic TAMs, pro-tumorigenic TAMs had higher intracellular lipid content and expression of ER stress-related proteins, including spliced X-box binding protein 1 (sXBP1), which was inducible by secreted factors in tumor cell-conditioned media. While XBP1-deficient bone marrow-derived macrophages (BMDMs) had reduced lipid accumulation and suppressive abilities when stimulated with tumor cell conditioned media, overexpression of sXBP1 alone in control-treated BMDMs did not result in increased pro-tumorigenic gene expression. Further analyses revealed that tumor-secreted factors induced inositol-requiring enzyme 1 (IRE1) to activate both STAT3 and XBP1 signaling to promote pro-tumorigenic TAMs. The group identified tumor-secreted β-glucosylceramide as a mediator of ER stress via macrophage inducible Ca2+-dependent lectin receptor (Mincle)-sensing, leading to increased cholesterol synthesis and a low phosphatidylcholine (PC) to phosphatidylethanolamine (PE) ratio. The resulting decrease in ER membrane fluidity was shown to ultimately cause activation of the IRE1-XBP1 and IRE1-STAT3 pathways to promote a pro-tumorigenic TAM phenotype. Interruption of this altered lipid composition by genetic and pharmacological modulation of lysophosphatidylcholine acyltransferase 3 (LPCAT3), the enzyme that synthesizes PC, prevented conditioned media-induced ER stress, expression of sXBP1, and shift to the pro-tumorigenic phenotype in BMDMs. In mouse models, treatment with liver X receptor agonist GW3965 to increase PC synthesis led to reduced tumor growth and fewer sXBP1-expressing immature TAMs.
Why this matters: This study delineates an important metabolic pathway involved in the generation of immunosuppressive tumor microenvironments and demonstrates a possible therapeutic strategy to reduce the numbers of intratumoral suppressive cells.
“Anti-Inflammatory Drugs Remodel the Tumor Immune Environment to Enhance Immune Checkpoint Blockade Efficacy” by Victoria S Pelly et al - Cancer Discovery 2021 11:2602-2619. doi: 10.1158/2159-8290.CD-20-1815
Victoria S Pelly and colleagues build on previous work showing that genetic modulation of the COX2/PGE2 pathway results in a spontaneous, favorable shift in the tumor microenvironment (TME) to show that selectively targeting this pathway with anti-inflammatory drugs enhances efficacy of checkpoint blockade. In CT26 colorectal cancer-bearing mice, the combination of the NSAID celecoxib plus anti-PD-1, methylprednisolone/prednisolone (MP+P) plus anti-PD-1, and MP+P plus anti-PD-1 plus anti-CTLA-4 all demonstrated improved tumor control and survival compared to anti-PD-1 alone. The addition of MP+P to anti-PD-1 resulted in identical leukocyte composition but reduced transcript levels of COX2-associated inflammatory mediators compared to checkpoint blockade monotherapy. Using a bilateral surgery model, mice were identified as “responders” with upregulated cytotoxic T cell (Cd8a, Gzmd, Ifng) and interferon gamma-related genes versus “non-responders” with upregulated protumorigenic genes. Once separately clustered, a clear pattern of enrichment for CD8+ cytotoxic T and NK cell populations emerged in the responders. The addition of celecoxib to anti-PD-1 was associated with increased interferon gamma-related gene expression and reduced PGE2/EP4 expression. Combination treatment with PGE2/EP4 inhibitors (EPAT) and anti-PD-1 led to complete tumor rejection, with a distinct interferon-related response signature compared to the other treatment schemes investigated. Culturing resected patient tumors with celecoxib ex vivo increased CXCL9 and CXCL10, dampened IL6, IL10, CXCL1, and CXCL5, and increased T-cell effector cytokines and activation markers.
Why this matters: Anti-inflammatory drugs induced an interferon-related genes signature in mice and are associated with enhanced T cell effector function in human tumors ex vivo. COX2/EPAT inhibition therefore could paradoxically represent a strategy to overcome resistance to checkpoint blockade.
“In vivo CRISPR screens identify the E3 ligase Cop1 as a modulator of macrophage infiltration and cancer immunotherapy target” by Xiaoqing Wang et al - Cell 184, 5357–5374, 2021 Oct 14
Macrophages contribute to an immunosuppressive tumor microenvironment (TME) that limits the efficacy of immune checkpoint blockade in several solid tumors, including triple negative breast cancer (TNBC) and colon cancer. Using in vivo CRISPR screens, Xiaoqing Wang and colleagues identified Cop1 as a macrophage regulator that contributes to the immunosuppression in the TME in murine TNBC and colon cancer models. A mouse lentiviral CRISPR-Cas9 (MusCK) library (>4,500 genes, 5 sgRNAs per gene) was transduced into 4T1 breast cancer cells, which were implanted into T cell deficient Foxn1nu/nu, immune competent, and ovalbumin-vaccinated BALB/c hosts. The primary screen identified 79 genes as differentially enriched for immune evasion in the competent hosts. A validation screen using 8 sgRNAS per gene focusing on the 79 genes identified during the first round identified Cop1 as having the greatest depletion in wild type versus nude hosts. Characterization of Cop1 knockout 4T1 tumors showed slower progression in untreated and anti-PD-1 treated hosts, downregulation of expression of transcripts for macrophage chemoattractants and activating cytokines (Ccl2, Ccl5, Ccl11, Ccl19, Ccl20, Cxcl4, Cxcl11, Gm-csf, and Il-6), decreased macrophage infiltration, and a shift favoring the M1 macrophage phenotype. Mass spectrometry revealed the transcription factor C/ebpδ as the only protein with Cop1-dependent degradation after treatment with the proteasome inhibitor MG132. CHiPseq showed that C/ebpδ chromatin occupancy was increased with knockout of Cop1, leading to differential expression of immune response and macrophage chemokine genes (Ccl2, Ccl7). Surprisingly, C/ebpδ lacks a conventional degron for Cop1 substrates. Trib2 was identified as a potential adaptor protein, and interaction between Cop1, Trib2, and C/ebpδ was confirmed by co-immunoprecipitation. Treatment with MG132 resulted in elevated protein levels of Trib2 and C/ebpδ, and polyubiquitination of C/ebpδ was decreased with Cop1 knockout. Furthermore, interrogation of human tumor datasets demonstrated an association between COP1 expression and the M2 macrophage phenotype, increased levels of macrophage-associated cytokines, and worse outcomes in several cancers.
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