BACKGROUND:Management of retroperitoneal liposarcoma (RPLPS) is challenging and recurrence rates remain high despite aggressive surgical resections. Preoperative radiation alone lacks definitive benefit, thus we sought to evaluate combined chemoradiotherapy with the potential to enhance local efficacy of radiation as well as control micrometastatic disease. We assessed the safety and tolerability of preoperative eribulin, a cytotoxic microtubule inhibitor approved for the treatment of advanced liposarcoma, in combination with radiation in patients with RPLPS. METHODS:In this open-label dose-finding study, patients with primary or recurrent resectable RPLPS received preoperative intensity-modulated radiation therapy (IMRT) with escalating doses of eribulin. Eribulin was administered for three 21-day cycles at a starting dose of 1.1 mg/m2. Concurrent radiation to 50.4 Gy began during cycle 1. Surgical resection occurred 3-10 weeks after completion of chemoradiation. The primary endpoint was determination of the recommended phase 2 doses (RP2D) of concurrent eribulin and radiation. RESULTS:Between 2018-2023, fifteen patients were enrolled. Thirteen patients were evaluable for dose-determination. Four patients treated at starting dose level had no dose-limiting toxicities (DLTs). Two of nine patients treated with escalated eribulin dose had DLTs. The RP2D was established as eribulin 1.4 mg/m2 and IMRT 50.4 Gy. Eleven patients were evaluable for secondary efficacy endpoints. The median recurrence-free survival was 30.4 months (95% CI 12.0-NR) and the median overall survival was 54.1 months (95% CI 9.5-NR). Patient reported outcome data did not show any significant changes over the study period. CONCLUSION:A preoperative chemoradiation protocol of eribulin in combination with IMRT showed a manageable safety profile and warrants additional prospective evaluation for treatment of resectable RPLPS.

Background & Aims:Tumor immune resistance is recognized as a contributor to low survivorship in pancreatic ductal adenocarcinoma (PDAC). The inflammatory cytokine interleukin-6 (IL-6) promotes polarization of CD4 T cell populations away from immune tolerance, induces differentiation of cytotoxic CD8 T cells, and drives expansion and anti-tumor activity in chimeric antigen receptor (CAR) T cell therapies. This work aims to test whether IL-6 could stimulate an anti-tumor response in PDAC. Methods:We overexpressed IL-6 in multiple KrasG12D/+, Tp53R172H/+, Pdx1-Cre (KPC) cell lines, which were orthotopically implanted in mice (OT-PDACIL6). We followed mouse survival and measured tumor growth, tumor histology, and plasma IL-6 at 5 and 10 days after tumor implantation. We measured tumor immune cell infiltration via flow cytometry and histology. We used antibody-based T cell depletion and secondary tumor implantation rechallenge to test the dependency of the durable immune reaction on T cells. Results:Improved survival occurred in all instances of OT-PDACIL6, with one cell line (KxPxCx) reproducibly resulting in long-term recurrence-free survival. With KxPxCx cells, circulating IL-6 was 100-fold higher in OT-PDACIL6 than in OT-PDACparental mice. Flow cytometry revealed increased T cells and NK cells, and decreased T regulatory cells, and we observed significantly increased lymphoid aggregates in OT-PDACIL6 as compared to OT-PDACparental tumors. Antibody-based CD4+ and CD8+ T cell depletion prevented tumor clearance and completely abolished the survival advantage in OT-PDACIL6 mice. The anti-tumor immune response to OT-PDACIL6 rendered mice immune to re-challenge with OT-PDACparental tumors. Conclusions:Locally high IL-6 concentrations potently enhance the T cell-mediated anti-tumor response to PDAC.

The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is characterized by a limited infiltration of tumor-specific T cells and anti-tumor T cell activity. Extracellular factors in the PDAC TME have been widely reported to mediate immune suppression, but contribution from tumor-intrinsic factors is not well understood. The RNA-binding protein, HuR (ELAVL1), is enriched in PDAC and negatively correlates with T cell infiltration. In an immunocompetent Kras-p53-Cre (KPC) orthotopic model of PDAC, we found that genetic disruption of HuR impaired tumor growth despite not impacting PDAC cell division. Importantly, we found that HuR depletion in tumors enhanced both T cell number and activation states. Mechanistically, HuR mediated stabilization of mTOR pathway transcripts, and inhibition of mTOR activity rescued the impaired function of local T cells. Phenotypically, we found that HuR induced T-cell suppression in PDAC, as HuR depletion sensitize PDAC tumors to immune checkpoint blockade, while isogenic, wildtype tumors are resistant. Our findings describe a novel role of HuR in facilitating tumor immune suppression in the PDAC TME by inhibiting T cell infiltration and function, and implicate HuR inhibition as a potential therapeutic combination with immunotherapy. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Immunotherapies have shown limited effectiveness in pancreatic ductal adenocarcinoma (PDAC), despite successes in many other cancers. The RNA-binding protein HuR is known to play a critical role in the oncogenesis of PDAC through regulating key mRNA transcripts. Our lab has showed that tumor-intrinsic HuR is overexpressed in PDAC and regulates the tumor microenvironment composition and alters multiple tumor cytokine release. Thus, we hypothesized that HuR plays a role in immune evasion of PDAC. We disrupted the locus encoding HuR (Elavl1), using CRISPR Cas9 in Kras-p53 mutant-driven (KPC) murine PDAC to assess its function in tumor immune evasion. Here, we report that HuR, a known pro-survival factor for PDAC, has a role in tumor immune evasion by suppressing T cell infiltration and T cell activation. Specifically, HuR-knockout (KO) KPC tumors grew slower compared to KPC wildtype (WT) tumors in an orthotopically implantation model (p-value<0.0001), even though they grew at the same rate in vitro (in a non-compromised environment). Accordingly, HuR-KO tumors had more T cell infiltration and activated T cells comparing to HuR-WT tumors (p-value=0.0047). Furthermore, T cell depletion partially rescued the size of HuR-KO tumors. Collectively, these data support the hypothesis that HuR suppresses T cell activation and prevents T cells from infiltrating the PDAC microenvironment, which therefore leads to PDAC immune evasion. Ongoing experiments will transduce KPC cell lines with ovalbumin (OVA) antigen, which will selectively active CD8+ OT-I T cells through their transgenic TCR, to measure the ability of KPC HuR-WT or HuR-KO OVA cells in directly activating CD8+ OT-I T cells. We will also rescue HuR in HuR-KO cells to validate HuR’s role in immune evasion. These findings support the concept that HuR plays an important pro-survival role of PDAC in vivo, and that targeting strategies against HuR being developed (e.g., siHuR nanotherapies and small molecule inhibitors) could potentially enhance PDAC sensitivity to immune-based anti-cancer therapies such as checkpoint blockade and T cell transfer. Citation Format: Yifei Guo, Jennifer M. Finan, Madeline D. Hedberg, Jonathan R. Brody, Robert Eil. The tumor-intrinsic RNA binding protein HuR is essential for anti-tumor immunity in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A043.

Supplemental Figure 1. The Na+/K+ ATPase prevents T cell mTOR and ROS associated transcriptional programs Experimental schema depicting isolation, electroporation with sgRNA + spCas9 complexes, and activation of CD8+ T cells with attendant gene set enrichment analysis (GSEA) comparing the abundance of transcripts making up the indicated gene sets in Atp1a1 deficient vs sufficient CD8+ T cells. Net enrichment score, NES. False discovery rate, FDR.

Abstract Immunotherapies have shown limited effectiveness in pancreatic ductal adenocarcinoma (PDAC), despite having success in other cancers. The RNA-binding protein HuR is known to play a critical role in the oncogenesis of PDAC by regulating key mRNA transcripts. Our lab has shown that tumor-intrinsic HuR is overexpressed in PDAC and regulates the tumor microenvironment composition, altering multiple tumor cytokines to release. Thus, we hypothesized that HuR plays a role in immune evasion of PDAC. We disrupted the locus encoding HuR (Elavl1) using CRISPR Cas9 in Kras-p53 mutant-driven (KPC) murine PDAC to assess its function in tumor immune evasion. Here, we report that HuR, a known pro-survival factor for PDAC, has a role in tumor immune evasion by suppressing T cell infiltration and T cell activation. Specifically, HuR-knockout (KO) KPC tumors grew slower compared to KPC wildtype (WT) tumors in an orthotopically implanted model (p-value < 0.0001), even though human PDAC cell lines with HuR-WT and HuR-KO grew at the same rate in NRG mice (in an immune-compromised environment). Accordingly, HuR-KO tumors had more T cell infiltration and activated T cells compared to HuR-WT tumors (p-value = 0.0047). Importantly, T cell depletion (both CD4+ and CD8+) partially rescued the size of HuR-KO tumors. Next, we rescued the expression of HuR in the HuR-KO cell line, and the re-expression partially rescued the tumor size in vivo. This observation validates a HuR-mediated dependent mechanism. Collectively, these data support the hypothesis that HuR suppresses T cell activation and prevents T cells from infiltrating into the PDAC microenvironment, leading to PDAC immune evasion. Ongoing experiments will transduce KPC cell lines with ovalbumin (OVA) antigen, which will selectively activate CD8+ OT-I T cells through their transgenic TCR, to measure the ability of KPC HuR-WT or HuR-KO OVA cells in directly activating CD8+ OT-I T cells. We will also investigate HuR's impact on immune cells' cytokine release and how that will impact the cytotoxicity of T cells. These findings support the ongoing concept that HuR plays an important pro-survival role in PDAC in vivo, and targeting strategies against HuR being developed (e.g., siHuR nano-therapies and small molecule inhibitors) could potentially enhance PDAC sensitivity to immune-based anti-cancer therapies, such as checkpoint blockade and T cell transfer. Citation Format: Yifei Guo, Jennifer M. Finan, Alexandra Q. Bartlett, Jonathan R. Brody, Robert Eil. The tumor-intrinsic RNA binding protein HuR is essential for anti-tumor immunity in PDAC [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 5357.

BACKGROUND:Most patients treated with the standard dosing protocol (SDP) of hepatic arterial infusion (HAI) floxuridine require dose holds and reductions, thereby limiting their HAI therapy. We hypothesized that a modified dosing protocol (MDP) with a reduced floxuridine starting dose would decrease dose holds, dose reductions, and have similar potential to convert patients with unresectable colorectal liver metastases (uCRLM) to resection. PATIENTS AND METHODS:We reviewed our institutional database of patients with uCRLM treated with HAI between 2016 and 2022. In 2019, we modified the floxuridine starting dose to 50% (0.06 mg/kg) of the SDP (0.12 mg/kg). We compared treatment related outcomes between the SDP and MDP cohorts. RESULTS:Of n = 33 patients, 15 (45%) were treated on the SDP and 18 (55%) with our new institutional MDP. The MDP cohort completed more cycles before a dose reduction (mean 4.2 vs. 2), received more overall cycles (median 7.5 vs. 5), and averaged 39 more days of treatment (all P < 0.05). The SDP experienced more dose reductions (1.4 vs. 0.61) and dose holds (1.2 vs. 0.2; both P < 0.01). Of the patients in each group potentially convertible to hepatic resection, three patients (23%) in the SDP and six patients (35%) in the MDP group converted to resection (P = 0.691). Overall, four patients (27%) in the SDP developed treatment ending biliary toxicity compared with one patient (6%) in the MDP. CONCLUSIONS:A 50% starting dose of HAI floxuridine provides fewer treatment disruptions, more consecutive floxuridine cycles, and a similar potential to convert patients with initially uCRLM for disease clearance.

511 Background: Most patients with intrahepatic cholangiocarcinoma (ICC) have unresectable or multifocal liver-dominant disease. Survival after first-line systemic therapy remains poor, with median progression-free (mPFS) and overall survival of 7.2 and 12.8 months, respectively. Hepatic arterial infusion (HAI) therapy with floxuridine maximizes liver-specific treatment with minimal systemic toxicity and potentially offers improved disease control when combined with systemic therapy. Methods: HELIX-1 (NCT04251715) is an investigator-initiated, first-line, single-center, single-arm phase II clinical trial for patients with liver-dominant unresectable or multifocal ICC. The trial was designed with a patient safety run-in evaluating toxicity from the combined therapy. Pre-trial screening included laparoscopy, biopsies, and PET/CT. Eligible patients were treated with systemic mFOLFIRINOX for 4 cycles in order to select those likely to benefit from HAI. Patients with disease control on restaging proceeded to HAI pump placement and treatment with HAI floxuridine for 14 days followed by systemic mFOLFIRI on a 28-day cycle. The co-primary objectives were to assess safety of the combined therapeutic strategy and the disease control rate (DCR) at 6 months (RECIST v1.1) at end of trial (EOT). Results: A total of n=5 patients with liver-only ICC enrolled in the trial and completed the entire study protocol. The median age was 60 years (range 42-69) with a dominant lesion size of 9.8cm (range 8.4-14.5). All patients had both right and left hemiliver involvement with a median of 9 intrahepatic tumors (range 1-15). No patients experienced grade 3 or 4 adverse events, or hepatic dysfunction leading to cessation of HAI therapy. The DCR at 6 months was 100%, with a mPFS of 18.2 months. All five patients achieved partial radiographic response (PR) while receiving HAI therapy, and remain alive with liver-only disease at a median of 18.4 months (range 12.7-20) after study enrollment. After continuing treatment with HAI and systemic mFOLFIRI beyond the EOT, two patients transitioned to HAI treatment only and then to biochemical and radiographic surveillance without therapy after demonstrating PR and CA19-9 normalization. Conclusions: Integration of HAI floxuridine with mFOLFIRI following mFOLFIRINOX induction for patients with liver-only advanced ICC is well-tolerated and demonstrates longer DCR in comparison to historical controls. The combined regimen minimized systemic toxicity and allowed a large proportion of patients to transition to liver-only HAI treatment with maintained disease control. Future directions include combining HAI with new first-line systemic regimens for patients with advanced ICC. Clinical trial information: NCT04251715 .

Abstract Tumor immune resistance contributes to low survivorship in patients with pancreatic ductal adenocarcinoma (PDAC). To understand how tumor-secreted factors might impact anti-tumor immunity, we developed a model of PDAC tumor overexpressing interleukin-6 (IL-6), a known driver of pancreatic tumorigenesis and cancer cachexia, in orthotopically implanted PDAC cancer cells (OT-PDACIL6). This model utilized codon-optimized IL6 to accurately detect tumor cells in the pancreas and distal organs via qPCR. OT-PDACIL6 tumors developed poorly- differentiated, infiltrative carcinomas, which were histologically similar to the PDAC cell line they were derived from (OT-PDACparental). Surprisingly, OT-PDACIL6 tumors were present at 5 days post implantation, but undetectable by histology or qPCR by 10 days post implantation. OT- PDACIL6 and sham mouse survival were equivalent (87.5%) at 76 days post-implantation. OT- PDACparental mortality was 100% at day 13. We concluded that OT-PDACIL6 tumors resolve spontaneously. IL-6 is well-established as a driver of cachexia, and we saw that OT-PDACIL6 tumors induced a rapid cachexia phenotype that began to resolve as the tumor was cleared. Plasma IL-6 levels tracked with tumor burden. At 5 days, plasma IL-6 was 100-fold higher in OT-PDACIL6 mice than in OT-PDACparental mice, but undetectable in OT-PDACIL6 mice after 12 days. We hypothesized that high levels of IL-6 were driving an anti-tumor immune response. To assess changes in tumor immune infiltrate that could lead to tumor clearance, we measured a broad range of immune cells using flow cytometry and immunofluorescent histology. At peak tumor burden, we observed exacerbated splenomegaly, and increased tumor-infiltrating T cells and NK cells in OT-PDACIL6 mice. T regulatory cells were decreased in OT-PDACIL6 tumors. CD4/CD8 antibody-based depletion prevented tumor clearance and significantly decreased survival of OT-PDACIL6 mice, indicating that T cells are necessary for IL-6-driven PDAC tumor clearance. We then tested whether the anti-tumor T cell response was durable using a secondary tumor challenge with PDACparental cells. This revealed that the anti-tumor T cell response elicited by mice bearing OT-PDACIL6 tumors was sufficient to prevent development of OT-PDACparental tumors, likely due to induction of long-term memory T cells during the initial tumor challenge. We assessed the effect of the anti-tumor immune response on metastases and found that PDACIL6 cells were detected by qPCR in lungs of 58% of mice at day 5, and not detected at day 12. We conclude that extremely high levels of tumor-derived IL-6 are sufficient to drive an anti-tumor T cell response that rapidly and durably clears the tumor. The combination of increased tumor-infiltrating T cells and decreased T regulatory cells are likely both important aspects of the immune response observed in OT-PDACIL6 mice. This surprising finding highlights the pleiotropic effects of IL-6, which is known to promote tumorigenesis and cachexia, and yet, in this work, is the driving force underlying tumor clearance. Citation Format: Paige C Arneson-Wissink, Alexandra Q Bartlett, Heike Mendez, Xinxia Zhu, Jessica Dickie, Matthew McWhorter, Peter R Levasseur, Parham Diba, Katelyn T Byrne, Gregory D Scott, Robert Eil, Aaron J Grossberg. Durable T-cell-mediated anti-tumor immune response to pancreatic cancer cells overexpressing interleukin 6 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B044.

Supplemental Figure 3. K+ limits T cell exhaustion and TCR-induced ROS generation

AbstractBackgroundPatients with pancreatic ductal adenocarcinoma (PDAC) often suffer from cachexia, a wasting syndrome that significantly reduces both quality of life and survival. Although advanced cachexia is associated with inflammatory signalling and elevated muscle catabolism, the early events driving wasting are poorly defined. During periods of nutritional scarcity, the body relies on hepatic ketogenesis to generate ketone bodies, and lipid metabolism via ketogenesis is thought to protect muscle from catabolizing during nutritional scarcity.MethodsWe developed an orthotopic mouse model of early PDAC cachexia in 12‐week‐old C57BL/6J mice. Murine pancreatic cancer cells (KPC) were orthotopically implanted into the pancreas of wild‐type, IL‐6−/−, and hepatocyte STAT3−/− male and female mice. Mice were subject to fasting, 50% food restriction, ad libitum feeding or ketogenic diet interventions. We measured longitudinal body composition by EchoMRI, body mass and food intake. At the endpoint, we measured tissue mass, tissue gene expression by quantitative real‐time polymerase chain reaction, whole‐body calorimetry, circulating hormone levels, faecal protein and lipid content, hepatic lipid content and ketogenic response to medium‐chain fatty acid bolus. We assessed muscle atrophy in vivo and C2C12 myotube atrophy in vitro.ResultsPre‐cachectic PDAC mice did not preserve gastrocnemius muscle mass during 3‐day food restriction (−13.1 ± 7.7% relative to food‐restricted sham, P = 0.0117) and displayed impaired fatty acid oxidation during fasting, resulting in a hypoketotic state (ketogenic response to octanoate bolus, −83.0 ± 17.3%, P = 0.0328; Hmgcs2 expression, −28.3 ± 7.6%, P = 0.0004). PDAC human patients display impaired fasting ketones (−46.9 ± 7.1%, P < 0.0001) and elevated circulating interleukin‐6 (IL‐6) (12.4 ± 16.5‐fold increase, P = 0.0001). IL‐6−/− PDAC mice had improved muscle mass (+35.0 ± 3.9%, P = 0.0031) and ketogenic response (+129.4 ± 44.4%, P = 0.0033) relative to wild‐type PDAC mice. Hepatocyte‐specific signal transducer and activator of transcription 3 (STAT3) deletion prevented muscle loss (+9.3 ± 4.0%, P = 0.009) and improved fasting ketone levels (+52.0 ± 43.3%, P = 0.018) in PDAC mice. Without affecting tumour growth, a carbohydrate‐free diet improved tibialis anterior myofibre diameter (+16.5 ± 3.5%, P = 0.0089), circulating ketone bodies (+333.0 ± 117.6%, P < 0.0001) and Hmgcs2 expression (+106.5 ± 36.1%, P < 0.0001) in PDAC mice. Ketone supplementation protected muscle against PDAC‐induced atrophy in vitro (+111.0 ± 17.6%, P < 0.0001 myofibre diameter).ConclusionsIn early PDAC cachexia, muscle vulnerability to wasting is dependent on inflammation‐driven metabolic reprogramming in the liver. PDAC suppresses lipid β‐oxidation and impairs ketogenesis in the liver, which is reversed in genetically modified mouse models deficient in IL‐6/STAT3 signalling or through ketogenic diet supplementation. This work establishes a direct link between skeletal muscle homeostasis and hepatic metabolism. Dietary and anti‐inflammatory interventions that restore ketogenesis may be a viable preventative approach for pre‐cachectic patients with pancreatic cancer.

Supplemental Figure 2. NAC constrains the function of wild-type and Atp1a1 deficient CD8 T cells

Supplemental figure 4. NAC provision alone is insufficient to rejuvenate Atp1a1 deficient T cell function in vivo

Abstract T cells genetically engineered to express a chimeric antigen receptor (CAR) specific for the molecule CD19 have achieved significant gains in the treatment of chemotherapy-resistant leukemia and lymphoma. Despite evidence of long-lived cures in patients with metastatic solid cancer after receiving tumor-specific T cells, no CAR-T cell treatment is currently approved for use against any solid cancer. A large majority of solid cancers express one of many tumor-associated antigens (TAAs) that are vulnerable to CAR T cell recognition. However, in nearly all cases those same TAAs are present in healthy tissues. Since CAR T cells cannot differentiate between cancer and normal cells, CAR T cell recognition of TAAs on healthy tissues provokes dose-limiting ‘on-target off-tumor’ toxicities at sites of endogenous expression (lung, intestine). Thus, there remains a pressing need to develop strategies to mitigate off-tumor toxicity of CAR T cells. To this end, we develop two immunocompetent models of CAR T cell transfer targeting TAAs abundant in metastatic colorectal cancer and pancreatic cancer. TAA-specific CAR T cells affected tumor shrinkage along with infiltrating and damaging sites of endogenous expression (primarily lung, pancreas, & colon). In both of our models, off-tumor toxicities were antigen-dependent and resulted in tissue damage, systemic inflammation, and host death that was titratable to CAR-T cell dose. TAA-specific CAR T cells infiltrated sites of endogenous expression such as the intestine, pancreas, and lungs. Notably, the respiratory system was the site of greatest pathology in both of our models, with massive CAR T cell accumulation and tissue destruction that resulted in systemic wasting and rapid weight loss. Remarkably, we found that the presence of an antigen-bearing tumor in the liver, but not other sites, greatly exacerbated CAR-T cell pulmonary infiltration, tissue destruction, and host mortality.The liver is the most common site of metastasis in patients with colorectal and pancreatic adenocarcinoma and the only site of metastasis in one-third of colorectal cancers. Our models indicated that TAA-specific CAR-T cells given for the treatment of liver metastasis result in dose-limiting pulmonary toxicity due to the presence of the TAA in the healthy lung. To address this translational barrier, we designed and validated a multiparameter genetic reprogramming targeting T cell trafficking and tissue residency introduced with the CAR to imbue CAR-T cells with liver tropism. In multiple models, CAR T cells programmed with liver-specific tropism augmented CAR T cell liver trafficking, decreased lung infiltration and toxicity, and effectively treated TAA+ liver metastasis. We aim to initiate a pilot clinical trial using tissue resident-programmed CAR T cells to treat colorectal liver metastasis. Citation Format: Alex Bartlett, Matthew Mcwhorter, Camille Collier, Chelsea Jenkins, Robert Eil. A novel approach to limit off-tumor toxicity of CAR-T cells targeting solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB344.

Abstract T cells are often compromised within cancers, allowing disease progression. We previously found that intratumoral elevations in extracellular K+, related to ongoing cell death, constrained CD8+ T-cell Akt–mTOR signaling and effector function. To alleviate K+-mediated T-cell dysfunction, we pursued genetic means to lower intracellular K+. CD8+ T cells robustly and dynamically express the Na+/K+ ATPase, among other K+ transporters. CRISPR-Cas9–mediated disruption of the Atp1a1 locus lowered intracellular K+ and elevated the resting membrane potential (i.e., Vm, Ψ). Despite compromised Ca2+ influx, Atp1a1-deficient T cells harbored tonic hyperactivity in multiple signal transduction cascades, along with a phenotype of exhaustion in mouse and human CD8+ T cells. Provision of exogenous K+ restored intracellular levels in Atp1a1-deficient T cells and prevented damaging levels of reactive oxygen species (ROS), and both antioxidant treatment and exogenous K+ prevented Atp1a1-deficient T-cell exhaustion in vitro. T cells lacking Atp1a1 had compromised persistence and antitumor activity in a syngeneic model of orthotopic murine melanoma. Translational application of these findings will require balancing the beneficial aspects of intracellular K+ with the ROS-dependent nature of T-cell effector function. See related Spotlight by Banuelos and Borges da Silva, p. 6

Colorectal cancer is the second leading cause of cancer-related deaths in the United States and accounts for an estimated 1 million deaths annually worldwide. The liver is the most common site of metastatic spread from colorectal cancer, significantly driving both morbidity and mortality. Although remarkable advances have been made in recent years in the management for patients with colorectal cancer liver metastases, significant challenges remain in early detection, prevention of progression and recurrence, and in the development of more effective therapeutics. In 2017, our group held a multidisciplinary state-of-the-science symposium to discuss the rapidly evolving clinical and scientific advances in the field of colorectal liver metastases, including novel early detection and prognostic liquid biomarkers, identification of high-risk cohorts, advances in tumor-immune therapy, and different regional and systemic therapeutic strategies. Since that time, there have been scientific discoveries translating into therapeutic innovations addressing the current management challenges. These innovations are currently reshaping the treatment paradigms and spurring further scientific discovery. Herein, we present an updated discussion of both the scientific and clinical advances and future directions in the management of colorectal liver metastases, including adoptive T-cell therapies, novel blood-based biomarkers, and the role of the tumor microbiome. In addition, we provide a comprehensive overview detailing the role of modern multidisciplinary clinical approaches used in the management of patients with colorectal liver metastases, including considerations toward specific molecular tumor profiles identified on next generation sequencing, as well as quality of life implications for these innovative treatments.