Sepsis increases perioperative metastases in a murine model

Abstract Background Cancer surgery can promote tumour metastases and worsen prognosis, however, the effect of perioperative complications on metastatic disease remains unclear. In this study we sought to evaluate the effect of common perioperative complications including perioperative blood loss, hypothermia, and sepsis on tumour metastases in a murine model. Methods Prior to surgery, pulmonary metastases were established by intravenous challenge of CT26LacZ colon cancer cells in BALB/c mice. Surgical stress was generated through partial hepatectomy (PH) or left nephrectomy (LN). Sepsis was induced by puncturing the cecum to express stool into the abdomen. Hemorrhagic shock was induced by removal of 30% of total blood volume (i.e. stage 3 hemorrhage) via the saphenous vein. Hypothermia was induced by removing the heating apparatus during surgery and lowering core body temperatures to 30 °C. Lung tumour burden was quantified 3 days following surgery. Results Surgically stressed mice subjected to stage 3 hemorrhage or hypothermia did not show an additional increase in lung tumour burden. In contrast, surgically stressed mice subjected to intraoperative sepsis demonstrated an additional 2-fold increase in the number of tumour metastases. Furthermore, natural killer (NK) cell function, as assessed by YAC-1 tumour cell lysis, was significantly attenuated in surgically stressed mice subjected to intraoperative sepsis. Both NK cell-mediated cytotoxic function and lung tumour burden were improved with perioperative administration of polyI:C, which is a toll-like receptor (TLR)-3 ligand. Conclusions Perioperative sepsis alone, but not hemorrhage or hypothermia, enhances the prometastatic effect of surgery in murine models of cancer. Understanding the cellular mechanisms underlying perioperative immune suppression will facilitate the development of immunomodulation strategies that can attenuate metastatic disease

Correction: Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence.

[This corrects the article DOI: 10.1371/journal.pone.0155947.]

Perioperative influenza vaccination reduces postoperative metastatic disease by reversing surgery-induced dysfunction in natural killer cells.

Purpose: Surgical removal of solid primary tumors is an essential component of cancer treatment. Surgery-induced dysfunction in natural killer (NK) cells has been linked to the development of metastases in animal models and patients with cancer. We investigated the activation of NK cells using influenza vaccine in the perioperative period to eradicate micrometastatic disease. Experimental design: Both the B16lacZ and 4T1 tumor models in immunocompetent mice were used to assess the in vivo efficacy of perioperative influenza vaccine administration. In healthy human donors and cancer surgery patients, we assessed NK cell function pre- and post-influenza vaccination using both in vivo and ex vivo assays. Results: Using the TLR3 agonist poly(I:C), we showed as proof-of-principle that perioperative administration of a nonspecific innate immune stimulant can inhibit surgery-induced dysfunction in NK cells and attenuate metastases. Next, we assessed a panel of prophylactic vaccines for NK cell activation and determined that inactivated influenza vaccine was the best candidate for perioperative administration. Perioperative influenza vaccine significantly reduced tumor metastases and improved NK cytotoxicity in preclinical tumor models. Significantly, IFNα is the main cytokine mediator for the therapeutic effect of influenza vaccination. In human studies, influenza vaccine significantly enhanced NK cell activity in healthy human donors and cancer surgery patients. Conclusion: These results provide the preclinical rationale to pursue future clinical trials of perioperative NK cell activation, using vaccination in cancer surgery patients. Research into perioperative immune therapy is warranted to prevent immune dysfunction following surgery and eradicate metastatic disease

Surgery-induced abrogation of protection conferred by AdDCT vaccination is dependent on CD3⁺ T cells.

<p><b>(a)</b> CD-1 nude mice were vaccinated with 1×10⁷ pfu AdDCT. On day 7, mice were challenged with sc B16F10lacZ tumors and then underwent surgery or no surgery. <b>(b)</b> Survival of treated B16F10lacZ tumor-bearing CD-1 nude mice are shown in Kaplan-Meier curves. Percentage of living mice is indicated. N = 7-8/group. <b>(c)</b> B6 mice were vaccinated with 1x10⁷ pfu AdDCT and at day 7, mice underwent surgery or no surgery. At day 8, spleen CD3⁺ T cells were isolated and transferred to naive recipient B6 mice. At day 9, recipient mice were challenged with sc B16F10lacZ tumors. <b>(d)</b> Survival of treated B16F10lacZ tumor-bearing mice are shown in Kaplan-Meier cures. Percentage of living mice is indicated. N = 7-8/group.</p

Surgical stress results in a decrease in CD8⁺ T cell cytokine production in response to TAA.

<p>B6 mice received neoadjuvant vaccination with 1×10⁷ pfu AdDCT. At day 7, mice were challenged iv with 3x10⁵ of B16F10lacZ cells in order to establish syngeneic lung melanoma metastases, and then underwent surgery or no surgery. At day 8, mice were sacrificed and underwent spleen immune cell assessment. Percentage of DCT-specific <b>(a)</b> IFNγ⁺, <b>(c)</b> TNFα⁺, <b>(d)</b> Granzyme B⁺ CD8⁺ T cells reacting to DCT_180-188 peptide exposure. <b>(b)</b> Representative flow cytometry dot plots of DCT-specific IFNγ⁺/CD8⁺ T-cells reacting to DCT_180-188 peptide exposure. <b>(e)</b> Quantification of SFU in IFNγ ELISpot assay. <b>(f)</b> Corresponding representative images of IFNγ ELISpot assay of CD8⁺ T-reacting to DCT_180-188 peptide exposure. (*P<0.05, **P<0.01, ***P<0.001).</p

Surgical stress impairs anti-tumor immunity resulting in lung metastasis and subcutaneous tumor growth.

<p><b>(a)</b> B6 mice were administered with neoadjuvant AdDCT at 1×10⁷ pfu. On day 7, mice were challenged iv with 3x10⁵ of B16F10lacZ cells in order to establish syngeneic lung melanoma metastases, and then underwent abdominal laparatomy and nephrectomy (Nx) or no surgery. <b>(b)</b> Lungs extracted at day 10 (3 days following tumor cell inoculation) from PBS, Nx, AdDCT, AdDCT+Nx and quantified by X-Gal staining. N = 5/group. <b>(c)</b> B6 mice were vaccinated with AdDCT, then challenged on day 7 with sc 1x10⁶ B16F10lacZ cells, then underwent surgery or no surgery. <b>(d)</b> Tumor volume of treated B16F10lacZ sc tumor bearing mice was monitored daily. <b>(e)</b> Survival of treated B16F10lacZ sc tumor-bearing mice are shown in Kaplan-Meier curves. Percentage of living mice is indicated. N = 7-8/group, (*P<0.05, ***P<0.001).</p

Surgically stressed T cells display reduced cytokine secretion, proliferation and tumor infiltration in response to TAA.

<p>B6 mice received neoadjuvant vaccination with 1×10⁷ pfu AdDCT. At day 7, mice were challenged iv with 3x10⁵ of B16F10lacZ cells in order to establish syngeneic lung melanoma metastases, and then underwent surgery or no surgery. At day 8, mice were sacrificed and underwent spleen immune cell assessment. <b>(a)</b> Total number of CD8⁺ T cells, <b>(b)</b> total number of Annexin V^-/7-AAD^-/CD8⁺ T (live), Annexin V⁺/7-AAD^-/CD8⁺ T (early apoptosis), Annexin V⁺/7-AAD⁺/CD8⁺ T (late apoptosis) cells, <b>(c)</b> percentage of BrdU⁺/CD8⁺ T cells, <b>(d)</b> percentage and <b>(e)</b> representative dot plot of DCT-tetramer⁺/CD8⁺ T cells reacting to DCT_180-188 peptide exposure, <b>(f)</b> quantification of SFU from DCT-tetramer⁺/CD8⁺ T cells reacting to DCT_180-188 peptide exposure in IFNγ ELISpot assay. <b>(g)</b> Total number of CD8⁺/CD3⁺ T cells per mg of tumor from B6 mice challenged with sc B16F10lacZ tumors mixed with matrigel on day 7. On day 10, matrigel plugs were removed and assessed for immune cell infiltration by flow cytometry. N = 4-6/group. (*P<0.05, ***P<0.001).</p

Surgery impairs vaccine function in a therapeutic B16 melanoma model of minimal residual disease and can be rescued with preoperative IFNα.

<p><b>(a)</b> B6 mice were injected sc with 1x10⁶ B16F10lacZ cells. At day 7, mice were vaccinated with AdDCT. At day 14, tumors were resected (Res) with a 2mm margin +/- surgical stress (Res+Nx). <b>(b)</b> Survival of treated B16F10lacZ tumor-bearing mice are shown in Kaplan-Meier curves. N = 7-8/group. <b>c)</b> Preoperative treatment at day 10 with 1 high dose (10,000 IU/mouse) and at days 11 through 13 with 3 low doses (1000 IU/mouse) of recombinant mIFNα in MRD vaccination model. Survival of treated B16F10lacZ tumor-bearing mice are shown in Kaplan-Meier curves. N = 5-6/group.</p