Immunological effects of heated intraperitoneal chemotherapy can be augmented by thymosin α1.

BACKGROUND: Peritoneal metastases of colorectal carcinoma origin (PM-CRC) are treated by cytoreductive surgery and heated intraperitoneal chemotherapy (HIPEC). However, the majority of patients recur, calling for novel treatments. We explored the immunogenic changes induced by HIPEC and the possibility to use thymosin α1 (Tα1) as an immune-stimulatory agent. METHODS: We used an experimental murine model of PM-CRC combined with mitomycin (MMC)-based HIPEC. We determined immune cell infiltration into tumor metastases after HIPEC administration by means of immunohistochemistry, and determined immunogenic cell death signals in tumor cells by real-time polymerase chain reaction. RESULTS: Mice with PM-CRC treated by HIPEC had increased overall survival (OS) compared to sham-treated mice (median OS 22.8 vs 18.9 days, respectively; P < 0.001). HIPEC induced increased infiltration of CD4+, CD8+, CD68 + and CD20 + cells into omental and visceral metastases at a magnitude of 40-100 %. We searched for potential immune signals induced by HIPEC by determining its effects on known immunogenic cell death proteins (heat-shock protein [HSP]-70, HSP-90 and calreticulin). HIPEC significantly increased HSP-90 mRNA expression (2.37 ± 1.5 vs 1-fold change, P < 0.05). The OS of Tα1 treated mice significantly improved compared to HIPEC-treated mice (16.3 ± 0.8 vs 14.1 ± 0.6 days, respectively, P = 0.02) and vs sham (11.8 ± 0.8 days, P = 0.007). CONCLUSIONS: HIPEC induced immunogenic changes that led to increased immune cell infiltration. These changes were further augmented by Tα1 treatment. Future studies aimed at optimizing Tα1 treatment should focus upon the immune response it evokes.

PD-1 Blockade Combined with Heated Intraperitoneal Chemotherapy Improves Outcome in Experimental Peritoneal Metastases from Colonic Origin in a Murine Model.

BACKGROUND: Heated intraperitoneal chemotherapy (HIPEC) was shown to induce immunogenicity of peritoneal metastases from colorectal cancer (PM-CRC) by induction of immunogenic cell death. We aimed to explore whether the addition of a checkpoint inhibitor would augment the effect of HIPEC in an experimental murine model of PM-CRC. METHODS: PM-CRC was established in C57BL mice by intraperitoneal inoculation of MC38 colon cancer cells. HIPEC was administered using the closed technique with mitomycin C (MMC). Clinical and immunological parameters were compared between animals treated with HIPEC alone and those treated with HIPEC + anti-programmed death receptor-1 (aPD-1). RESULTS: MMC-based HIPEC increased the overall survival of animals compared with sham-treated animals (22.8; 95% confidence interval [CI] 21.14-24.53 vs. 18.9 days; 95% CI 17.6-20.3, p < 0.001). The extent of peritoneal disease as measured by the modified peritoneal carcinomatosis index was also reduced by HIPEC. This clinical benefit was accompanied by increased infiltration of CD8+, CD68+, and CD20+ cells into tumor metastases in HIPEC-treated animals compared with sham-treated animals. We identified heat shock protein (HSP) 90 as a potential immunogenic cell death protein whose expression is increased under HIPEC conditions (fold change: 2.37 ± 1.5 vs. 1 without HIPEC, p < 0.05). Combined HIPEC + PD-1 treatment ameliorated survival compared with HIPEC alone and sham treatment (24.66; 95% CI 20.13-29.2 vs. 19; 95% CI 15.85-22.14 and 14.33 days; 95% CI 9.6-19.04, respectively; p = 0.008). This clinical effect was accompanied by increased CD8+ tumor infiltration. CONCLUSIONS: HIPEC induced the expression of immunogenic cell death signals that can support an anti-tumor immune response. This response can be further exploited by a checkpoint inhibitor.

Thymosin alpha 1 as an adjuvant to hyperthermic intraperitoneal chemotherapy in an experimental model of peritoneal metastases from colonic carcinoma.

INTRODUCTION: Heated intraperitoneal chemotherapy (HIPEC) is currently implemented in the treatment of peritoneal metastases from colorectal carcinoma (PM-CRC) origin. However, recurrence is common and the effectiveness of HIPEC has been questioned. The aim of this study was to evaluate the use of thymosin alpha 1 (Tα1), an immunomodulatory molecule, as an adjuvant to HIPEC treatment. METHODS: We developed an experimental model of HIPEC by the induction of PM-CRC in C57BL mice and intra-abdominal perfusion of mitomycin C (MMC). Mice were treated with Tα1 at 0.6 mg/kg for 5 days after HIPEC. Clinical and immunological parameters were compared between HIPEC and HIPEC + Tα1 groups. RESULTS: Treatment with Tα1 increased overall survival of mice compared to HIPEC treatment alone and sham-treated animals (16.1 ± 0.8 vs. 14.1 ± 0.6 and 11.8 ± 0.8, respectively, p = 0.02). Tα1 had no direct anti-tumor effect, as seen by lack of inhibition of tumor cell proliferation. Tα1 treatment induced a T helper (Th) 1 immune response in tumor metastases as evidenced by a significant increase of the Th1-specific markers IFN-γ and T-bet (1.21 ± 0.3 vs. 0.52 ± 0.08, p < 0.05; 0.88 ± 0.04 vs. 0.64 ± 0.14, p < 0.05, respectively). This Th1 skew was accompanied by increased CD8+ infiltration into omental and visceral metastases by Tα1 treatment compared to sham and HIPEC-treated animals (21.24 ± 2.16 vs. 10.45 ± 0.89 and 7.7 ± 1.3, p < 0.001; 14.12 ± 1.54 vs. 12.12 ± 0.01 and 6.64 ± 0.87, p < 0.01, respectively). CONCLUSIONS: Tα1 augments the effect of HIPEC by the induction of a Th1 anti-tumor immune response. Further experiments should evaluate Tα1 and other novel immunomodulators in order to exploit the immunological opportunities created by HIPEC.

Protective Desmoplasia in Pancreatic Adenocarcinoma: High Vitamin D Receptor Expression and Collagen Content.

BACKGROUND/AIM: Vitamin D receptor (VDR) has been shown to suppress desmoplasia in pancreatic ductal adenocarcinoma (PDAC). Our aim was to assess the clinical effects of VDR expression and its correlation with collagen content in the desmoplasia of PDAC patients. PATIENTS AND METHODS: This is a retrospective analysis of 127 patients with peritumoral desmoplasia resected for PDAC. VDR expression and collagen content were assessed by immunohistochemistry and correlated with clinical outcome. RESULTS: Patients were classified into those with high and those with low VDR expression. High VDR expression was associated with improved overall survival (OS) in localized disease (N0) (median= 33; 95%CI=26.4-39.6 vs. 18; 15.5-20.5 months, p=0.01). Patients with high vs. low collagen content had improved OS [34, (range=22.3-45.6 months) vs. 17, (range=14.4-19.6 months), p<0.001]. The number of VDR+ cells was the same for patients with either high or low collagen content. CONCLUSION: Protective desmoplasia is associated with increased VDR expression and collagen content.

Size and lipid modification determine liposomal Indocyanine green performance for tumor imaging in a model of rectal cancer.

Localization of rectal tumors is a challenge in minimally invasive surgery due to the lack of tactile sensation. We had developed liposomal indocyanine green (Lip-ICG) for localization of rectal tumor. In this study we evaluated the effects of liposome size and lipid PEGylation on imaging. We used an endoscopically-guided orthotopic experimental rectal cancer model in which tumor fluorescence was determined at different time points after intravenous (i.v.) administration of Lip-ICG and PEGylated liposomes (PEG-Lip-ICG). Signal intensity was measured by tumor-to-background ratio (TBR), or normalized TBR (compared to TBR of free ICG). Fluorescence microscopy of tumor tissue was performed to determine fluorescence localization within the tissue and blood vessels. Liposomes of 60 nm showed an increased TBR compared with free ICG at 12 hours after i.v. injection: normalized TBR (nTBR) = 3.11 vs. 1, respectively (p = 0.006). Larger liposomes (100 nm and 140 nm) had comparable signal to free ICG (nTBR = 0.98 ± 0.02 and 0.78 ± 0.08, respectively), even when additional time points were examined (0.5, 3 and 24 hours). PEG-Lip- ICG were more efficient than Lip-ICG (TBR = 4.2 ± 0.18 vs. 2.5 ± 0.12, p < 0.01) presumably because of reduced uptake by the reticulo-endothelial system. ICG was found outside the capillaries in tumor margins. We conclude that size and lipid modification impact imaging intensity.

Desmoplasia in Lymph Node Metastasis of Pancreatic Adenocarcinoma Reveals Activation of Cancer-Associated Fibroblasts Pattern and T-helper 2 Immune Cell Infiltration.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a peritumoral proliferation of fibroblasts and extracellular matrix production known as desmoplasia. We aimed to study desmoplasia in PDAC lymph node (LN) metastases. METHODS: We evaluated LNs from 66 patients with PDAC and LN metastases. We used immunohistochemistry and real-time polymerase chain reaction to phenotype the desmoplastic response. RESULTS: Desmoplasia was identified in 57% of patients with LN metastases (Des+). Cancer-associated fibroblasts (CAFs) in Des+ expressed α-smooth muscle actin and collagen 11A1. The latter expression was present only in CAFs but not in LN stroma or in LN metastases without desmoplasia (Des-). Desmoplasia was associated with upregulation of transforming growth factor ß messenger RNA. Whereas numbers of CD8+ in tumor vicinity were not different between Des+ and Des- patients (78 [standard deviation {SD}, 57] vs 92 [SD, 52], P = 0.48, respectively), the numbers of GATA-3+ cells, a marker of T-helper 2 immune response was significantly increased (3.7 [SD, 6.3] for Des+ vs 1.3 [SD, 2.7] for Des-, P < 0.05). CONCLUSIONS: Lymph node desmoplasia is associated with CAF pattern activation and Th2 infiltration. Therapeutic modulation of desmoplasia may be relevant in the metastatic phase and influence antitumor immune response.

T-Helper 1 Immune Response in Metastatic Lymph Nodes of Pancreatic Ductal Adenocarcinoma: A Marker For Prolonged Survival.

BACKGROUND: Although lymph node (LN) metastases is considered a grave prognostic sign in pancreatic ductal adenocarcinoma (PDAC), patients with positive lymph nodes (PLN) constitute a heterogeneous group. Our purpose was to identify morphological and immune parameters in the primary tumor and in PLN of resected PDAC patients, which could further stratify these patients to different subgroups. METHODS: We retrospectively evaluated histological and immunohistochemical characteristics of 66 patients with PDAC who were operated at our institution. These were subsequently correlated to clinical outcome. RESULTS: Mean patient age and number of LN harvested was 65.5 ± 10.3 and 12.3 ± 6.5 years, respectively. Tumor size (T stage) and perineural invasion had no effect on clinical outcome. High-grade tumor was associated with decreased survival [overall survival (OS) = 19.6 ± 2.7 months for poorly differentiated PDAC vs. 31.2 ± 4 for well and moderately differentiated, p = 0.03]. Patients with ≥ 8 PLN had significantly worse outcome (OS = 7.3 ± 0.8 months for PLN ≥ 8 vs. OS = 30.1 ± 3.2 months for PLN < 8, p < 0.0001). T helper (Th) 1 immune response was measured both by its effector cells (CD8+) and expression of its main transcription factor, T-bet. CD8+ high patients had significantly increased OS compared with CD8+ low (OS = 36.8 ± 5.3 months for CD8 + high vs. OS = 24.3 ± 3.5 for CD8 + low, p = 0.03) Similarly, Th1 predominant immune response measured by T-bet expression was associated with improved OS compared with non-Th1 (OS = 32.8 ± 3.2 vs. OS = 19.5 ± 2.9, p < 0.0001). CONCLUSIONS: Our data indicate an association between Th1-type immune response and increased survival. Future research is needed to exploit Th1 immune response as a biological marker for immunotherapy.

Intraoperative Localization of Rectal Tumors Using Liposomal Indocyanine Green.

BACKGROUND: Tumor localization may pose a significant challenge during minimally invasive rectal resection. Near-infrared (NIR) imaging can penetrate biological tissue and afford tumor localization from the external surface of the rectum. Our aim was to develop an NIR-based tool for rectal tumor imaging that can be administered intravenously. METHODS: We prepared indocyanine-green (ICG)-loaded liposomes by sonication. Liposomes were evaluated for their size and morphology. We then used an endoscopically induced rectal cancer in mice as a model for rectal cancer. After intravenous administration, tumors were evaluated for their fluorescence intensity. Tumor intensity was expressed in relation to the background signal, that is, tumor to background ratio (TBR). RESULTS: Liposomes in various sizes could be prepared by adjusting sonication time. We selected 100-nm-sized liposomes for further experiments. Transmission electron microscopy showed spherical particles and confirmed the size measurements. The liposomes could be lyophilized and then rehydrated again before use without compromising their structure or signal. Fluorescence intensity was kept for 24 hours after solubilization. Testing the optimal time course for rectal tumor imaging revealed that early time course (up to 3 hours) yielded nonspecific imaging, whereas after long time course (24 hours), a very weak signal remained in the tissue. The optimal time window for imaging was after 12 hours from injection, with TBR = 8.1 ± 3.6 ( P = .002). Free ICG could not achieve similar results. CONCLUSIONS: The liposomal ICG can be reproducibly prepared and kept in lyophilized form. Liposomal ICG could serve as a tool for intraoperative tumor localization.

Analysis of histological and immunological parameters of metastatic lymph nodes from colon cancer patients reveals that T-helper 1 type immune response is associated with improved overall survival.

Lymph node (LN) involvement in colonic carcinoma (CC) is a grave prognostic sign and mandates the addition of adjuvant treatment. However, in light of the histological variability and outcomes observed, we hypothesized that patients with LN metastases (LNM) comprise different subgroups.We retrospectively analyzed the histological sections of 82 patients with CC and LNM. We studied various histological parameters (such as tumor grade, desmoplasia, and preservation of LN architecture) as well as the prevalence of specific peritumoral immune cells (CD8, CD20, T-bet, and GATA-3). We correlated the histological and immunological data to patient outcome.Tumor grade was a significant prognostic factor even in patients with LNM. So was the number of LN involved (N1/N2 stage). From the morphological parameters tested (LN extracapsular invasion, desmoplasia in LN, LN architecture preservation, and mode of metastases distribution), none was found to be significantly associated with overall survival (OS). The mean OS of CD8 low patients was 66.6 ±â€Š6.25 versus 71.4 ±â€Š5.1 months for CD8 high patients (P = 0.79). However, T-helper (Th) 1 immune response skewing (measured by Th1/Th2 ratio >1) was significantly associated with improved OS. For patients with low ratio, the median OS was 35.5 ±â€Š5 versus 83.5 months for patients with high Th1/Th2 ratio (P = 0.001).The histological presentation of LNM does not entail specific prognostic information. However, the finding of Th1 immune response in LN signifies a protective immune response. Future studies should be carried to verify this marker and develop a strategy that augments this immune response during subsequent adjuvant treatment.