Macrophage Migration Inhibitory Factor protects cancer cells from immunogenic cell death and impairs anti-tumor immune responses

<div><p>The Macrophage Migration Inhibitory Factor (MIF) is an inflammatory cytokine that is overexpressed in a number of cancer types, with increased MIF expression often correlating with tumor aggressiveness and poor patient outcomes. In this study, we aimed to better understand the link between primary tumor expression of MIF and increased tumor growth. Using the MMTV-PyMT murine model of breast cancer, we observed that elevated MIF expression promoted tumor appearance and growth. Supporting this, we confirmed our previous observation that higher MIF expression supported tumor growth in the 4T1 murine model of breast cancer. We subsequently discovered that loss of MIF expression in 4T1 cells led to decreased cell numbers and increased apoptosis <i>in vitro</i> under reduced serum culture conditions. We hypothesized that this increase in cell death would promote detection by the host immune system <i>in vivo</i>, which could explain the observed impairment in tumor growth. Supporting this, we demonstrated that loss of MIF expression in the primary tumor led to an increased abundance of intra-tumoral IFNgamma-producing CD4+ and CD8+ T cells, and that depletion of T cells from mice bearing MIF-deficient tumors restored growth to the level of MIF-expressing tumors. Furthermore, we found that MIF depletion from the tumor cells resulted in greater numbers of activated intra-tumoral dendritic cells (DCs). Lastly, we demonstrated that loss of MIF expression led to a robust induction of a specialized form of cell death, immunogenic cell death (ICD), <i>in vitro</i>. Together, our data suggests a model in which MIF expression in the primary tumor dampens the anti-tumor immune response, promoting tumor growth.</p></div

Macrophage Migration Inhibitory Factor protects cancer cells from immunogenic cell death and impairs anti-tumor immune responses - Table 1

<p>Macrophage Migration Inhibitory Factor protects cancer cells from immunogenic cell death and impairs anti-tumor immune responses</p> - Table

MIF expression in the primary tumor leads to decreased dendritic cell abundance and activation in the tumor.

<p>1.0 x 10⁴ WT or MIF KD 4T1 cells were implanted in the mammary fat pad of female Balb/c mice. <b>A,</b> Tumors were harvested and weighed at day 8 of tumor growth, which is the first point at which palpable tumors are detectable. Tumors were digested and analyzed by flow cytometry for infiltration of dendritic cells by cell surface markers <b>(B)</b> and activation markers <b>(C)</b>. Representative flow plots are shown in panels B and C. n = 6 mice per group. One-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001.</p

MIF expression in the primary tumor dampens anti-tumor T cell responses in vivo.

<p>1.0 x 10⁴ WT or MIF KD 4T1 cells were implanted in the mammary fat pad of female Balb/c mice. <b>A,</b> Tumors were harvested at day 10 of tumor growth and weighed. Tumors were then digested and dissociated, and cells were cultured <i>in vitro</i> for 4 hours in the presence of BFA +/- anti-CD3 stimulation. Cells were stained for CD4 and CD8 surface expression <b>(B)</b> and intracellular IFNgamma <b>(C)</b> by flow cytometry. Data shown are representative of one of three independent experiments, with n = 6 mice/group. One-way ANOVA. * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.</p

MIF expression promotes tumor progression in the MMTV-PyMT and 4T1 models of breast cancer.

<p>Female WT and MIF KO MMTV-PyMT mice were monitored starting at 6 weeks of age for the presence of mammary tumors. <b>A,</b> Age was recorded on the day of appearance of the first tumor. n = 19 for WT mice, n = 21 for MIF KO mice. <b>B-C,</b> Mice were euthanized at 8 weeks of age. Detectable tumor material and non-tumor bearing fat pads were removed, weighed, and enumerated. Each data point on dot plots represents one individual fat pad or tumor, with some mice having multiple tumors. n = 4 mice for WT and n = 3 mice for MIF KO. <b>D-F,</b> Mice were euthanized at 5 months of age. Tumors were removed from the fat pads, enumerated, and weighed. Each data point in <b>(D)</b> represents one mouse. Each data point in <b>(E)</b> represents one tumor. n = 8 mice for WT, n = 11 mice for MIF KO. <b>G,</b> 1.0 x 10⁴ WT or MIF KD 4T1 cells were implanted in the mammary fat pad of female Balb/c mice and tumor size was monitored starting at day 10 by caliper measurement. <b>H,</b> Tumors were harvested at day 22 of tumor growth post implantation and weighed. Data in <b>G</b> and <b>H</b> are representative of three independent experiments, with n = 5 mice/group in each experiment. One-way ANOVA. *p<0.05, ** p<0.01, *** p<0.001 **** p<0.0001.</p

Experimental model.

<p>Upon loss of MIF expression in the tumor, cancer cells undergo ICD. This leads to an increased abundance and activation of DCs in the tumor microenvironment, resulting in an enhanced T cell-mediated anti-tumor immune response through secretion of IFNgamma in the tumor microenvironment.</p

MIF-expressing tumor cells show decreased markers of immunogenic cell death under serum-free conditions.

<p><b>A-C,</b> WT or MIF KD 4T1 cells were grown in 10% serum-containing media overnight, and then media was replaced with fresh 10% serum or serum-free media. After 24 hours, media was sampled and tested for extracellular ATP. After 48 hours, cells were harvested and stained by flow cytometry for cell surface expression of calreticulin or HSP70. <b>D-F,</b> MIF KD 4T1 cells reconstituted (recon) with WT MIF, or P2G MIF, or with an empty vector as a control were grown in 10% serum-containing media overnight, and then switched to serum-free media. After 48 hours, media was sampled and tested for extracellular ATP and cells were harvested and stained by flow cytometry for cell surface expression of calreticulin or HSP70. Data shown are the means of 3 independent experiments, with 3 replicates per experiment. <b>A,</b> Student’s t-test, <b>B-F</b>, one-way ANOVA. * p<0.05, ** p<0.01.</p

MIF expression promotes cell growth and protects against cell death in vitro in serum-free conditions.

<p><b>A,</b> WT or MIF KD 4T1 cells were grown in 10% serum-containing media overnight, and then switched to fresh 10%, serum, 1% serum, or serum-free media. Cells were counted using a hemocytometer every day for 3 days. Determination of the slope of the lines using best-fit analysis of the curves during the exponential growth phase for each condition demonstrates that growth rate of the WT cells is greater than that of the MIF KD in both 1% serum (WT = 250417 +/- 11333 vs MIF KD = 181111 +/- 3836) and serum-free conditions (WT = 85000 +/- 3368 vs MIF KD = 53056 +/- 13391). Moreover, the mean cell number at the curve peak is higher in the WT cultures in both 1% (WT = 650000+/-20367 vs MIF KD = 497778+/-12814 at day 4) and serum free conditions WT = 200000+/-5774 vs MIF KD = 136111+/-14087 at day 3). <b>B,</b> WT or MIF KD 4T1 cells were grown in 10% serum-containing media overnight, and then switched to fresh 10% or serum-free media for a further 48 hours. Cells were then stained for Annexin V and PI using flow cytometry, or <b>C,</b> stained for cleaved caspase 3 by flow cytometry. <b>D,</b> MIF KD 4T1 cells were reconstituted (recon) with WT MIF, P2G MIF or with an empty vector as a control and were grown in 10% serum-containing media overnight, and then switched to fresh 10% media (top) or serum-free media (bottom) and counted using a hemocytometer every day for 3 days. Examination of the growth rate as in Panel A demonstrates that reconstitution with WT MIF (but not the P2G mutant) rescued both the growth rate (+WT Recon = 140676 vs +P2G Recon = 108000, +Vector Recon = 107491) and the peak cell number (+WT Recon = 311352+/-27168 vs +P2G Recon = 246000+/-13443, +Vector Recon = 244981+/-18604) in serum free growth conditions. <b>E,</b> After 48 hours in fresh 10% or serum-free media, cells were stained for Annexin V and PI (left) or cleaved caspase 3 (right) using flow cytometry. Data is the mean of 3 independent experiments with 3 replicates per experiment. One-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001.</p

SFN suppresses the HIF-1–mPGES –PGE2 axis by control of HIF-1α protein expression without altering mRNA levels.

<p>A, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 4 hours. Total RNA was analyzed by quantitative RT-PCR. The results are expressed as fold change relative to untreated cells (mean ± SEM; n = 3). B&C, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added or not and cells were cultured for another 4 hours. Total cell lysates were subjected to immunoblot analysis (panel B) and total RNA was analyzed by quantitative RT-PCR (panel C).</p

SFN does not inhibit COX-2 expression in A549 cells.

<p>A, A549 cells were pre-treated with or without different concentrations of SFN for 30 minutes. IL1β (1 ng/ml) was then added (left) or not (right) and cells were cultured for another 24 hours. Total cell lysates were subjected to immunoblot analysis. B, A549 cells were pretreated with or without SFN for 30 minutes. IL1β (1 ng/ml) was then added and cells were cultured for another 4 hours. Total RNA was analyzed by RT-PCR. The results are expressed as fold change relative to untreated cells (mean ± SEM; n = 3). C, A549 cells were treated with 1 ng/ml IL1β either with or without 10 µM SFN for different times. Total cell lysates were subjected to immunoblot analysis.</p