Macrophage subtypes inhibit breast cancer proliferation in culture

Macrophage subtypes inhibit breast cancer proliferation in culture | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Macrophage subtypes inhibit breast cancer proliferation in culture Sophia R.S. Varady , Daniel Greiner , View ORCID Profile Minna Roh-Johnson doi: https://doi.org/10.1101/2024.06.01.596963 Sophia R.S. Varady 1 Department of Biochemistry, University of Utah School of Medicine; Salt Lake City , UT, 84112, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daniel Greiner 1 Department of Biochemistry, University of Utah School of Medicine; Salt Lake City , UT, 84112, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Minna Roh-Johnson 1 Department of Biochemistry, University of Utah School of Medicine; Salt Lake City , UT, 84112, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Minna Roh-Johnson For correspondence: roh-johnson{at}biochem.utah.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Macrophages are a highly plastic cell type that adopt distinct subtypes and functional states depending on environmental cues. These functional states can vary wildly, with distinct macrophages capable of displaying opposing functions. We sought to understand how macrophage subtypes that exist on two ends of a spectrum influence the function of other cells. We used a co-culture system with primary human macrophages to probe the effects of macrophage subtypes on breast cancer cell proliferation. Our studies revealed a surprising phenotype in which both macrophage subtypes inhibited cancer cell proliferation compared to cancer cells alone. Of particular interest, using two different proliferation assays with two different breast cancer cell lines, we showed that differentiating macrophages into a “pro-tumor” subtype inhibited breast cancer cell proliferation. These findings are inconsistent with the prevailing interpretation that “pro-tumor” macrophages promote cancer cell proliferation and suggest a re-evaluation of how these interpretations are made. Introduction Macrophages are a component of the innate immune system, known for their role in killing microorganisms and removing dead cells. What is perhaps less known is that macrophages are a highly flexible cell type found in almost all tissues (for review, see ( Mosser and Edwards, 2008 )). Distinct macrophage differentiation states lead to their functional diversity, in which macrophages regulate a variety of processes including development ( Eom and Parichy, 2017 ), cancer progression ( Lin and Pollard, 2004 ; Nielsen and Schmid, 2017 ), and repair of blood vessels ( Liu et al ., 2016 ; Gurevich et al ., 2018 ). Thus, macrophages are a unique cell type, in that an individual cell can transition between many different phenotypic states within its lifetime ( Azizi et al ., 2018 ; Locati et al ., 2020 ; Cheng et al ., 2021b ). Macrophages are often classified into two major subtypes representing two distinct ends of an activation spectrum: M1-activated or M2-activated ( Ginhoux et al ., 2016 ; Shapouri-Moghaddam et al ., 2018 ; Yunna et al ., 2020 ; Boutilier and Elsawa, 2021 ). M1-like macrophages are classically activated, pro-inflammatory macrophages, known for their role in eliminating pathogens and their anti-tumorigenicity ( Sica and Mantovani, 2012 ; Martinez and Gordon, 2014 ; Pan et al ., 2020 ). Conversely, M2-like macrophages are anti-inflammatory and function in tissue healing and tumor growth ( Mantovani et al ., 2002 ; Shapouri-Moghaddam et al ., 2018 ; Boutilier and Elsawa, 2021 ). Both macrophage subtypes are typically classified by their surface marker expression, but these two classifications are simplistic and recent studies have revealed variation in the expression of these markers ( Ginhoux et al ., 2016 ; Muller et al ., 2017 ; Azizi et al ., 2018 ). M2-like macrophages are representative of macrophages in the tumor ( Hughes et al ., 2015 ; Steenbrugge et al ., 2018 ). Macrophages are abundant in many solid tumors and promote cancer progression ( Lin and Pollard, 2004 ; Chen et al ., 2005 ; Obeid et al ., 2013 ; Mantovani et al ., 2017 ; Nielsen and Schmid, 2017 ; Hinshaw and Shevde, 2019 ; Pan et al ., 2020 ; Xiang et al ., 2021 ). The existing paradigm is that M1-like macrophages inhibit cancer growth, and M2-like macrophages promote cancer growth ( Zhang et al ., 2014 ; Liu et al ., 2021 ; Zhang and Sioud, 2023 ). However, there are a number of factors that complicate interpretations. These conclusions are based on comparisons of cancer cell proliferation in culture with M1-like versus M2-like macrophages ( Sousa et al ., 2015 ; Moraes et al ., 2017 ; Zhang et al ., 2023a ), but do not reveal how macrophage subtypes regulate cancer cell proliferation compared to cancer cells alone ( Sousa et al ., 2015 ; Moraes et al ., 2017 ; Zhang et al ., 2023a ). These comparisons are critical to interpret how macrophages ultimately regulate cancer cell proliferation. Furthermore, it is important to note that most of the reported studies use macrophage conditioned media approaches ( Mu et al ., 2018 ; Deng et al ., 2021 ; Zhang et al ., 2021 ; Chen et al ., 2022 ; Cheng et al ., 2023 ; Nie et al ., 2023 ). While informative, these studies do not address how cell contact between macrophages and cancer cells regulate outcomes. Finally, many approaches use macrophage cell lines (THP-1, U937 and RAW 264.7) which are cancer cells themselves ( Chanput et al ., 2014 ; Taciak et al ., 2018 ; Nascimento et al ., 2022 ), and there is a dearth of studies using physiologically relevant macrophages. Given that macrophages adopt differentiation states that represent two ends of a spectrum, we sought to use this simplified system to fundamentally understand how different macrophage subtypes regulate the behavior of another cell. By comparing macrophage/cancer cell co-cultures to cancer cells alone, and taking advantage of a direct co-culture system, as well as the commonly employed conditioned media system, we made a surprising observation in which the presence of either M1-like or M2-like macrophages inhibit cancer cell proliferation compared to cancer cells alone. These results are in contrast to interpretations made from previous studies, and provide a previously undescribed function for M2-like macrophages in breast cancer proliferation. Results and Discussion M2-like macrophages do not promote cancer cell proliferative capacity Previous literature has shown that macrophages differentiated into an M2-like subtype promote cancer cell proliferation when compared to macrophages differentiated into an M1-like subtype ( Liu et al ., 2020 ; Zhao et al ., 2021 ; Zhang et al ., 2023a ). These findings are consistent with data generated in vivo , in which tumor-associated macrophages are tumor-promoting ( Dandekar et al ., 2011 ; Cendrowicz et al ., 2021 ; Munir et al ., 2021 ), although we recognize that the complexity of tumor-associated macrophages is not recapitulated with in vitro macrophage polarization studies ( Jia et al ., 2016 ; Ramesh et al ., 2020 ; Ma et al ., 2022 ). Nevertheless, these findings have put forth a model by which M2-like macrophages promote cancer cell proliferation. To understand how macrophage subtypes regulate cancer cell proliferation compared to cancer cells alone, we used an in vitro system using primary human macrophages ( Figure 1A ). We isolated primary monocytes from healthy blood donors, and differentiated them into either an M1-like or M2-like macrophage state using previously published approaches ( Jia et al ., 2016 ; Zheng et al ., 2018 ; Little et al ., 2019 ; Mohd Yasin et al ., 2022 ; Kidwell et al ., 2023 ) for subsequent experiments. Download figure Open in new tab Figure 1. MDA-MB-231 breast cancer cells do not exhibit increased proliferation when in culture with M2-like macrophages. (A) Schematic outlining co-culture approaches. (B) Example gating of Ki67 cell cycle index flow cytometry assays. (C-F) Quantification of MDA-MB-231 cells in each stage of the cell cycle at 48 (C) and 72 (E) hours, following gating strategies outlined in (B) when MDA-MB-231 cells are plated alone (“231 mono”), when MDA-MB-231 cells are plated with M1-like macrophages (“231 + M1 MΦ”) and when MDA-MB-231 cells are plated with M2-like macrophages (“231 + M2 MΦ”) (n=4 independent donors). (D, F) Comparison of only the G2/M phase of the cell cycle from data in (C) at 48 hours and data in (E) at 72 hours (n=4; Tukey’s 1-way ANOVA). (G) Representative example of MDA-MB-231 cell confluence over 72 hours when MDA-MB-231 cells are plated under conditions described in (C), plus MDA-MB-231 cells plated alone at double density (“231 double density”). (H) Quantification of MDA-MB-231 cell confluence at 72 hours in (G), reported as the fold increase in cell area over time (n=4 independent donors; Tukey’s 1-way ANOVA). M1-like or M2-like macrophages were plated in direct co-culture with a breast cancer cell line, MDA-MB-231 (231 cells), stably expressing a histone-localized mCherry (H2B-mCherry) ( Figure 1A ). 231 cells are a breast cancer line that lacks estrogen, progesterone, and HER2 receptors, and is thus referred to as “triple negative breast cancer”, which is a type of breast cancer with few treatment options and poor prognosis. We first used a flow cytometry assay to evaluate 231 proliferation changes by quantifying the presence of a proliferation marker Ki67 ( Endl and Gerdes, 2000 ; Kim and Sederstrom, 2015 ), as well as DNA content. With this approach, we quantified the percentage of 231 cells within the G2 and Mitotic (M) phases of the cell cycle ( Figure 1B ). When comparing 231 proliferative capacity in the presence of M1-like macrophages versus cancer cells alone, 231 cells exhibited a significantly lower percentage of cells in the G2/M phase of the cell cycle, which is consistent with previous reports showing “anti-tumor” activity of M1-like macrophages ( Figure 1, C-F ) ( Jia et al ., 2016 ; Ramesh et al ., 2020 ; Moradi-Chaleshtori et al ., 2021 ). However, quite surprisingly, when comparing the percentage of cancer cells within the G2/M phase of the cell cycle when in co-culture with M2-like macrophages versus cancer cells alone, we also observed a significant decrease in cancer cell proliferation when cancer cells were cultured with M2-like macrophages at 48 hours ( Figure 1, C-D ), which continued to trend at 72 hours ( Figure 1, E-F ). These results were surprising given that the prevailing model is that M2-like macrophages promote cancer cell proliferation ( Liu et al ., 2020 ; Chen et al ., 2021 ; Zhang et al ., 2023a ). The increase in the population of cancer cells within the G2/M phase of the cell cycle could reflect increased proliferation or a G2/M cell cycle arrest. Thus, we took advantage of an assay that quantifies cell confluence over time ( Figure 1, G-H ). Consistent with the cell cycle results, we observed a significant decrease in cancer cell proliferation when cancer cells are co-cultured with either M1-like or M2-like macrophages ( Figure 1, G-H ; Figure S1A; Supplemental Video S1) compared to cancer cells alone. To rule out the effects of cell confluence, we plated cancer cells at the same number as that used in co-cultures (231 mono) as well as double the number (231 double density) and found no difference in cancer cell proliferation between these two conditions ( Figure 1, G-H ). Furthermore, we asked whether the culture system may be masking a proliferation phenotype if the cancer cells are already dividing at maximum capability. Thus, we performed the same experiments in lower serum and first found that the basal proliferation rate of 231 cells was reduced when cells were cultured in 0.5% and 2% serum compared to full serum (10%) (Figure S1B). Using these low serum conditions, consistent with our previous results, M2-like macrophages in both 0.5% and 2% serum still inhibited breast cancer cell proliferation compared to cancer cells alone at 72 hours (Figure S1, C-D). These results suggest that M2-like macrophages do not promote breast cancer cell proliferation. M2-like macrophages retain their M2 status over time Due to the surprising results that M2-like macrophages do not promote cancer cell proliferation compared to cancer cells alone, we sought to confirm the differentiation status of the macrophages. We hypothesized that the lack of cancer cell proliferation in the presence of M2-like macrophages could be due to macrophages losing their M2-like state over time (see methods). We performed flow cytometry analysis of CD206 expression (M2-like marker) ( Figure 2, A-C ; Figure S2, A-B) as has been done previously ( Shrivastava et al ., 2019 ; Li et al ., 2024 ; Tang et al ., 2024 ; Zhang et al ., 2024 ). M2-like macrophages in co-culture with 231 cells maintain expression of CD206 from 24-72 hours at a similar level to control (M2-like macrophages alone) ( Figure 2, A-C ) We also quantified M1-like macrophage status over time using CD80 (M1-like marker). We found that M1-like macrophages exhibited higher levels of CD80/CD206 expression at 24, 48 and 72 hours compared to M2-like macrophages as has been done previously ( Ramesh et al ., 2020 ) (Figure S2, C-F). These results suggest that both M1-like and M2-like macrophages retain their differentiation status during the course of the experiments. Download figure Open in new tab Figure 2. M2-like macrophages maintain differentiation state over the experimental time course and do not induce MDA-MB-231 cell death. (A-C) Quantification of CD206+ macrophages across culture conditions. Quantification of the percent of CD206+ macrophages measured by surface CD206 expression at 24 hours (A), 48 hours (B), and 72 hours (C) of culture when M1-like macrophages are plated alone (“M1 MΦ”), when M1-like macrophages are plated with MDA-MB-231 cells (“231 + M1 MΦ”), when M2-like macrophages are plated alone (“M2 MΦ”), and when M2-like macrophages are plated with MDA-MB-231 cells (“231 + M2 MΦ”) (n=4 independent donors; Tukey’s 1-way ANOVA). (D-F) Quantification of Annexin V positive MDA-MB-231 cells at 24 hours (D), 48 hours (E), and 72 hours (F) when MDA-MB-231 cells are plated as described in (A-C). The positive control is heat-killed cells (see methods) (n=3 independent donors; Tukey’s 1-way ANOVA). Analysis was performed on the same day; thus, the same positive control was used for all time points. Lack of cancer cell proliferation with M2-like macrophages is not due to increased cancer cell death or spreading We next determined whether the reduced breast cancer cell number observed with M2-like macrophages was due to reduced cancer cell proliferation or increased cancer cell death. We quantified cancer cell death via Annexin V staining via flow cytometry ( Figure 2, D-F ; Figure S3, A-C). Annexin V detects phosphatidylserine, which is indicative of early stages of apoptosis ( van Engeland et al ., 1998 ). We found that at 24 hours, M1-like macrophages increased 231 cell death compared to 231 cells alone and M2-like macrophage co-culture ( Figure 2D ). At 48 and 72 hours, cancer cells in culture with M1-like macrophages exhibited subtle, but non-significant, increases in cancer cell death compared to cancer cells alone and cancer cells co-cultured with M2-like macrophages ( Figure 2, E-F ). This increase in cancer cell death in the presence of M1-like macrophages is consistent with previous reports that M1-like macrophages can be cytotoxic( Italiani and Boraschi, 2014 ; Shapouri-Moghaddam et al ., 2018 ; Najafi et al ., 2019 ). However, the level of cancer cell death observed in this condition (8.3% at 72 hours) does not account for the decreased cancer cell proliferation observed in our cancer cell + M1-like macrophage co-cultures (37% decrease) ( Figure 1H ). Furthermore, 231 cells in co-culture with M2-like macrophages did not show differences in cell death compared to 231 cells alone ( Figure 2, D-F ), suggesting the observed decrease in cancer cell proliferation in the presence of M2-like macrophages is not due to cell death. We measured proliferation in the time courses by quantifying the amount of surface area on the plate taken up by cancer cells over time ( Figure 1, G-H ). Thus, it is possible that increases in overall surface area are due to increased cancer cell spreading and not increases in cell number. To test between these hypotheses, we measured individual cancer cell area at 12 hours (time of cell attachment) and 72 hours (final time point) (Figure S3, D-E). At 12 hours, we did not observe any differences in cancer cell area across conditions (Figure S3D). At 72 hours, cancer cells exhibited a smaller cell area when in co-culture with M1-like macrophages (Figure S3E); however, importantly, we did not observe a difference in cell area of cancer cells in culture with M2-like macrophages versus cancer cells alone. Taken together with our cell death analysis, these results support the model that cancer cell proliferation is inhibited in the presence of M2-like macrophages. M2-like macrophages do not promote cancer cell proliferation in MDA-MB-468 cells Our findings that M2-like macrophages do not promote breast cancer cell proliferation were unexpected. Thus, we tested this hypothesis in another triple negative breast cancer cell line, MDA-MB-468 (468 cells), under the same culture conditions. Using flow cytometry to quantify Ki67 and DNA content, M2-like macrophages did not promote 468 cell proliferation at 48 and 72 hours ( Figure 3, A-D ) compared to cancer cells alone. We then quantified 468 cell proliferation over time and observed a decrease when in the presence of M2-like macrophages compared to 468 cells alone ( Figure 3, E-F ), consistent with results we observed with 231 cells ( Figure 1H ). Thus, these data further support our finding that M2-like macrophages do not promote cancer cell proliferation. Download figure Open in new tab Figure 3. MDA-MB-468 cells do not show increased proliferation when in culture with M2-like macrophages. (A-D) Quantification of MDA-MB-468 cells in each stage of the cell cycle at 48 (A) and 72 (C) hours when MDA-MB-468 cells are plated alone (“468 mono”), when MDA-MB-468 cells are plated with M1-like macrophages (“468 + M1 MΦ”) and when MDA-MB-468 cells are plated with M2-like macrophages (“468 + M2 MΦ”) (n=4 independent donors). (B, D) Comparison of only the G2/M phase of the cell cycle from data in (A) at 48 hours and data in (C) at 72 hours (n=4; Tukey’s 1-way ANOVA). (E) Representative example of MDA-MB-468 cell confluence over 72 hours when MDA-MB-468 cells are plated as in (A), plus MDA-MB-468 cells are plated alone at double density (“468 double density). (F) Quantification of MDA-MB-468 cell confluence at 72 hours in (E), reported as the fold increase in cell area over time (n=4 independent donors; Tukey’s 1-way ANOVA). Conditioned media from M2-like macrophages do not promote cancer cell proliferation The discrepancy in our results compared to the field caused us to compare our approaches with conditioned media approaches used previously ( Mor et al ., 1998 ; Sousa et al ., 2015 ; Jedrzejewski et al ., 2020 ; Li et al ., 2020 ). We used conditioned media from either M1-like or M2-like macrophages alone (M1 mono CM or M2 mono CM), as well as conditioned media from macrophage/cancer cell co-cultures (ie. M1 + 231 CM or M2 + 231 CM), and asked how each of these conditioned media affects the proliferation of breast cancer cells. We found that conditioned media from M2-like macrophages alone ( Figure 4, A-B ) or 231 + M2 MΦ co-culture ( Figure 4C ) caused subtle increases in 231 cell proliferation versus M1 conditioned media, although not statistically significant. With 468 cells, we found that 468 cells in M2 + 468 conditioned media exhibited significantly increased proliferation compared to M1 + 468 conditioned media at 72 hours ( Figure 4, D-F ). Together these results suggest that when comparing the effects of M2-like macrophages on cancer cell proliferation to M1-like macrophages, we observe results that are consistent with those in the field ( Zhang et al ., 2021 ; Zhao et al ., 2021 ; Cheng et al ., 2023 ). Download figure Open in new tab Figure 4. Conditioned media from M2-like macrophages increases MDA-MB-468 proliferation compared to conditioned media from M1-like macrophages. (A) Representative example of proliferation of MDA-MB-231 cell confluence over 72 hours when naïve MDA-MB-231 cells are treated with M2-like macrophage conditioned media (“M2 mono CM”), when naïve MDA-MB-231 cells are treated with MDA-MB-231/M2-like macrophage co-culture conditioned media (“M2 + 231 CM”), when naïve MDA-MB-231 cells are treated with M1-like macrophage conditioned media (M1 mono CM), and when naïve MDA-MB-231 cells are treated with MDA-MB-231/M1-like macrophage co-culture conditioned media (“M1 + 231 CM”). (B) Quantification of MDA-MB-231 cell confluence at 48 hours when treated with M1-like or M2-like macrophage monoculture CM in (A) (n=4 independent donors; Mann-Whitney test). (C) Quantification of MDA-MB-231 cell confluence at 72 hours when treated with MDA-MB-231/M1-like or MDA-MB-231/M2-like macrophage co-culture conditioned media in (A) (n=5 independent donors; Mann-Whitney test). (D) Representative example of proliferation of MDA-MB-468 cell numbers over 72 hours when naïve MDA-MB-468 cells are treated as described in (A). (E) Quantification of MDA-MB-468 cell confluence at 48 hours when treated with M1-like or M2-like macrophage monoculture CM in (D) (n=5 independent donors; Mann-Whitney test). (F) Quantification of MDA-MB-468 cell confluence at 72 hours when treated with MDA-MB-468/M1-like or MDA-MB-468/M2-like macrophage co-culture conditioned media in (D) (n=5 independent donors; Mann-Whitney test). We next asked how macrophage conditioned media regulates cancer cell proliferation versus cancer cells alone. Additionally, we asked how these conditions compare to direct contact co-cultures. We first found that the presence of M1-like macrophages (either in direct co-cultures or conditioned media experiments) inhibited 231 cell proliferation compared to 231 cells alone ( Figure 5, A-B ), again consistent with previous reports of M1-like macrophage-induced cytotoxicity ( Engstrom et al ., 2014 ; Moradi-Chaleshtori et al ., 2021 ). Interestingly, M1 macrophage conditioned media inhibited cancer cell proliferation better than direct 231 + M1 MΦ co-cultures ( Figure 5, A-B ). However, we found that conditioned media from M2-like macrophages did not increase cancer cell proliferation compared to cancer cells alone ( Figure 5, C-D ), as previous literature has suggested ( Deng et al ., 2021 ; Moradi-Chaleshtori et al ., 2021 ; Cheng et al ., 2023 ; Nie et al ., 2023 ; Yu et al ., 2023 ; Zhang et al ., 2023a ). When we performed the same experiments with 468 cells, we observed results that were consistent with the 231 experiments ( Figure 5, E-H ). We again tested the effects of macrophage conditioned media on cancer cell proliferation under low serum conditions. In low serum conditions (0.5% and 2%), conditioned media from M2-like macrophages, either from M2-like macrophages alone or 231 + M2 MΦ co-cultures, did not promote 231 cell proliferation compared to 231 cells alone (Figure S4, A-B). Download figure Open in new tab Figure 5. Conditioned media from M2-like macrophages inhibit breast cancer cell proliferation compared to cancer cells alone. (A) Representative example of proliferation of MDA-MB-231 cell numbers over 72 hours when naïve MDA-MB-231 cells are treated with MDA-MB-231 monoculture conditioned media (“231 mono”), when MDA-MB-231 cells are in direct co-culture with M1-like macrophages (“231 + M1 MΦ”), when naïve MDA-MB-231 cells are treated with MDA-MB-231/M1-like macrophage co-culture conditioned media (“M1 mono CM”), and when naïve MDA-MB-231 cells are treated with MDA-MB-231/M1-like macrophage co-culture conditioned media (“M1 + 231 CM”). (B) Quantification of MDA-MB-231 cell confluence at 72 hours when plated in conditions listed in (A) (n=4 independent donors; Tukey’s 1-way ANOVA). (C) Representative example of proliferation of MDA-MB-231 cell numbers over 72 hours when naïve MDA-MB-231 cells are treated with conditions described in (A), but with M2 macrophages. (D) Quantification of MDA-MB-231 cell confluence at 72 hours when plated in conditions listed in (C) (n=4 independent donors; Tukey’s 1-way ANOVA). (E) Representative example of proliferation of MDA-MB-468 cell numbers over 72 hours when naïve MDA-MB-468 cells are treated with conditions described in (A). (F) Quantification of MDA-MB-468 cell confluence at 72 hours when plated in conditions listed in (E) (n=5 independent donors; Tukey’s 1-way ANOVA). (G) Representative example of proliferation of MDA-MB-468 cell numbers over 72 hours when naïve MDA-MB-468 cells are treated with conditions described in (B). (H) Quantification of MDA-MB-468 cell confluence at 72 hours when plated in conditions listed in (G) (n=5 independent donors; Tukey’s 1-way ANOVA). While many studies have investigated the role of macrophage subtypes on cancer progression, comparisons are typically made between macrophage subtypes, leading to conclusions that M2-like macrophages promote cancer cell proliferation ( Liu et al ., 2020 ; Cheng et al ., 2023 ; Hao et al ., 2023 ; Yu et al ., 2023 ). However, using direct co-culture systems, conditioned media approaches, and two proliferation assays, our study shows an unexpected phenotype in which M2-like macrophages do not promote breast cancer cell proliferation compared to breast cancer cells alone, and may, in fact, decrease breast cancer cell proliferation. These results raise several interesting points about how macrophages regulate cancer cell behavior, and how this process relates to cancer progression. The small number of studies that have compared cancer cell proliferation when in culture with macrophages versus cancer cells alone present mixed results, with studies showing that M2-like macrophages promote breast cancer cell proliferation ( Carroll et al ., 2016 ; Hao et al ., 2023 ), and others showing that M2-like macrophages inhibit breast cancer cell proliferation ( Lindsten et al ., 2017 ; Hirano et al ., 2023 ). Our work is consistent with the results from the latter studies, and suggest that the different results obtained from these experiments could be due to the type of macrophage (primary versus cell line), how the macrophages were differentiated, and the cancer cell type. Additional work is required to fully understand this process. There is a wealth of data showing that the presence of tumor-associated macrophages promote tumor growth and metastasis ( Cheng et al ., 2021a ; Kumari and Choi, 2022 ; Zhang et al ., 2023b ; Zhao et al ., 2024 ). How do we reconcile these results with our results showing that the presence of macrophages decreases breast cancer cell proliferation compared to cancer cells alone? In our study, we do not model tumor-associated macrophages since differentiating macrophages in culture will not fully recapitulate the macrophage differentiation status in a tumor. However, we used our system to fundamentally understand how “pro-tumor” M2-like macrophages versus “anti-tumor” M1-like macrophages specifically regulate cancer cell proliferation. Given that macrophage infiltration correlates with tumor grade, these results suggest that early in tumorigenesis ( Mahmoud et al ., 2012 ; Yuan et al ., 2019 ; Jamiyan et al ., 2020 ), cancer cells begin to proliferate in the absence of stromal cells. Indeed, macrophage infiltration is still an early step in breast cancer tumorigenesis ( Zhukova et al ., 2020 ; Zhu et al ., 2021 ), but we would argue that whether tumor growth is a direct effect of pro-tumor macrophages or a consequence of the function of pro-tumor macrophages on other stromal cells is unclear. Tumor-associated macrophages have been shown to promote tumor angiogenesis which brings nutrients into the tumor, fueling tumor growth ( Riabov et al ., 2014 ; Fu et al ., 2020 ). Tumor-associated macrophages also promote local cancer cell invasion and entry into the vasculature compared to cancer cells alone ( Roh-Johnson et al ., 2014 ; Harney et al ., 2015 ). Thus, it is possible that the growth observed in tumors with either exogenous M2-like macrophages added ( Cho et al ., 2012 ; Steenbrugge et al ., 2018 ) or differentiating endogenous macrophages into an M2-like status ( Tao et al ., 2020 ; Stachowicz-Suhs et al ., 2024 ) is due to effects on the tumor environment, rather than directly on cancer cells. It is also possible that the direct role of tumor-associated macrophages on breast cancer cells is reduced cancer cell proliferation to support local cancer invasion, a key step in metastasis. Thus, future work is required to tease apart the direct functions of macrophages on cancer cell behavior. Methods Cell culture of cell lines Human cell lines MDA-MB-231 (HTB-26) and MDA-MB-468 (HTB-132) were purchased from the American Type Culture Collection (ATCC) and cultured at 37°C with 5% CO 2 according to their recommendations. Base culture media used was DMEM (high-glucose; 11965118, ThermoFisher), and 10% heat-inactivated fetal bovine serum (FBS; F4135, ThermoFisher). All cell lines were kept in culture for no more than 20 passages total. The MDA-MB-468 cell line was supplemented with 1% sodium pyruvate. Mycoplasma testing was performed every 6 months using the Mycoplasma Detection Kit (30–1012 K, ATCC). Isolation and culture of PBMCs Leukocyte cone filters were obtained from Associated Regional and Clinical Pathologists (ARUP) laboratories from unidentified healthy human blood donors. CD14+ monocytes were isolated as described( Greiner et al ., 2022 ) with the exception of selection being adherence-based to isolate monocytes from buffy coats. Freshly harvested primary monocytes were plated at a density of 20-25 M per 10 cm plate in macrophage culture media containing: RPMI (11875119, ThermoFisher), 10% FBS (26140079, Thermo Fisher), 0.5% penicillin/streptomycin (P/S; P4333, Thermo Fisher), 10 mM HEPES (15630080, ThermoFisher), 0.1% 2-Mercaptoethanol (21985023, Thermo Fisher), and recombinant human GM-CSF at 20 ng/mL (300–03, Peprotech). The following day (day 1), additional macrophage media was added, and on day 4, macrophage media was replaced. On day 7, macrophages were differentiated into either M1 or M2 activation states, or remained undifferentiated as ‘M0’ like macrophages acting as an activation control. Macrophage activation by cytokines To differentiate macrophages into an M1-like state, IFN-γ (3000–02, Peprotech, 50 ng/mL) and lipopolysaccharide (LPS; 00-4976-03, eBioscience, 10 ng/mL) were added to macrophage media for 72 hours prior to experiments. For M2-like activation, IL-4 (200-04, Peprotech, 20 ng/mL) and IL-13 (200–13, Peprotech, 20 ng/mL) were added to macrophage media for 72 hours prior to experiments. Once plated for experiments, both M1-like and M2-like macrophage media did not contain cytokines (macrophage culture media + GM-CSF only) so as not to effect cancer cells with activation cytokines. Cloning and cancer cell transduction The pLenti6-H2B-mCherry plasmid was purchased from Addgene (89766). MDA-MB-231 and MDA-MB-468 cell lines were transduced with lentiviral plasmid vectors to create stable lines. First, HEK 293 FT cells were plated on poly-L-lysine-coated 15 cm dishes. HEK 293 FT cells were transfected with PEI-max (24765, Polysciences) and plasmids for pCMV-VSV-G, psPax2, and the H2B-mCherry transgene cassette. Cells were washed the following day and grown for an additional 36 hours in fresh media. The supernatants were harvested, passed through 0.45 μm filters, and used fresh as previously described ( Johnson et al ., 2020 ). Lentiviral supernatants were used to transduce cell lines by plating 50,000 cells into one well of a 6-well plate with the lentiviral supernatant, which was diluted 1:5 in DMEM media with a final concentration of 10 μg/mL polybrene (TR-1003-G, Sigma). After 48 hours, the cells were expanded and flow sorted using the BD FACS Aria cytometer to select for fluorescent expression. Ki67 cell cycle assay MDA-MB-231 and MDA-MB-468 cell lines, respectively, were cultured directly with distinct macrophage subtypes (M1-like or M2-like, as described in ‘Isolation and culture of PBMCs’). Co-cultures for either cancer cell type were always cultured in macrophage media + GM-CSF (20 ng/mL) without additional polarization cytokines so as not to perturb cancer cells. Co-culture samples were enzymatically detached using Trypl-E Express Enzyme (1x) (12605028, ThermoFisher). Samples were incubated with staining buffer containing anti-human BV711-CD11b at 1:20 for 30 min on ice before fixation with the eBioscience Foxp3/Transcription Factor Staining Buffer Set (00-5523-00, ThermoFisher), according to manufacturer’s instructions. Cells were then stained with an APC conjugated Ki-67 antibody (APC-Ki67; 17-5699-42, ThermoFisher) at 1:20 for 30 min followed by a 3 µM DAPI (D9542, Sigma) solution for 10 min. Cells were resuspended in cold DPBS for analysis on the Fortessa Flow Cytometer. Single color controls were included for compensation in flow analysis. Single cells were selected by DNA width and DNA area, as has been done previously for Ki67 assays ( Di Rosa et al ., 2021 ; Kidwell et al ., 2023 ), due to its increased specificity compared to standard single cell selection methods for flow cytometry. Cancer cells were distinguished from macrophages based on CD11b-negative controls and no color cancer cell controls. Gating strategies are outlined in Figure 1B . Incucyte cell proliferation assay and analysis The Sartorius Incucyte S3 was used to measure MDA-MB-231 and MDA-MB-468 cell proliferation in various conditions including direct co-culture with M1-like or M2-like activated macrophages, as well as conditioned media conditions. Cell conditions were plated in replicates of 6 per experimental run and imaged at 20x magnification every 4 hours over the course of 72 hours. Proliferation was determined by cell area at each imaging time point. Cell area was defined by phase images and parameters were manually adjusted to correctly define individual cells. Total cell area per well was averaged across technical replicate wells of each condition. Confluence was reported as fold increase in phase area normalized to the first time point. The final time point (72 hours) fold increase in area was compared across conditions to measure statistical differences in changes in confluence. In addition, results were normalized to the first time point to account for slight variation in initial plating densities. Since macrophages were in the experimental system, but are post-mitotic, we included “double density” cancer cell monoculture controls to account for additional cell numbers in co-culture conditions, and confirm that initial confluence did not affect our results. For conditioned media experiments, naïve cancer cells were plated at a density of 7,500 cells/well in monoculture conditions (this includes all CM experiments) in a 96-well plate. Cell spreading was assessed by measuring cell area of 231 cell monoculture cells, 231 cells in 231/M1-like macrophage co-cultures, and 231 cells in 231/M2-like macrophage co-cultures at 12 hours (when the cells first adhered to the plate) and 72 hours. Measurements were taken in ImageJ by manual cell selection and area measurement. Conditioned media experiments Conditioned media conditions included M1 macrophage monoculture conditioned media, M1 co-culture with 231 or 468 cells conditioned media, M2 macrophage monoculture conditioned media, and M2 co-culture with 231 or 468 cells conditioned media. For conditioned media experiments, macrophages were first plated in their respective activation media, before being switched into M0 media and either being co-cultured with cancer cells or remaining in culture alone for 24 hours (as per the conditions listed above). Macrophages and cancer cells were plated at a ratio of 2:1 in 6-well dishes with 1 mL of media. Conditioned media was collected from culture conditions indicated above and added to “naïve” cancer cells (previously untreated cancer cells) for 24 hours prior to the experiment. A conditioned media cancer cell monoculture control was included to account for potential nutrient depletion in conditioned media treatments. This control consisted of naïve cancer cells treated with cancer cell conditioned media (cancer cell monoculture media collected from 24 hour culture prior to experiment). Annexin V cell death assay Fluorescently labelled MDA-MB-231 cells (mito-mEmerald, Addgene, 185596) were cultured with M1 or M2 activated macrophages in direct co-culture in macrophage media + GM-CSF (20 ng/mL) without additional polarization cytokines. After co-culture for 24, 48 or 72 hours, cells were enzymatically dissociated using trypsin-EDTA (25200056, ThermoFisher) and resuspended in serum containing media. Full dead controls used in flow cytometry analysis were prepared by lifting cells by scraping, followed by heating at 55°C for 20 minutes. Dead cells were cooled on ice after incubation. Alive/dead controls of half alive macrophages/half dead macrophages and half alive cancer cells/half cancer cells were included for fluorescence minus one (FMO) - Annexin V controls to determine gates for Annexin V-positivity. Cell samples were centrifuged at 600G for 5 minutes, and pellets were resuspended in BV711-CD11B antibody (macrophage marker; BioLegend, 301344) suspended in flow buffer (1:20 dilution) for 30 minutes on ice in the dark. Following centrifugation, cells were resuspended in Annexin V antibody (A35122, ThermoFisher) diluted in 1X Annexin V binding buffer (556454, BD Biosciences). Annexin V binding buffer was used to dilute the antibody prior to centrifugation and resuspension in flow cytometry buffer. Flow cytometry was performed on the BD LSR Fortessa (5 Lasers: UV, 405, 488, 561, 640). Cancer cells were distinguished from macrophages based on mEmerald-expression. Macrophages were distinguished by BV711-CD11B (1:20)-positive staining. No color controls were included to establish cancer cell+ gating, which was applied to all samples. Gating strategies are outlined in Supplemental Figure 3. Macrophage polarization status by flow cytometry M1 and M2 activated macrophages were plated in monoculture or co-culture with MDA-MB-231 cells in macrophage media + GM-CSF (20 ng/mL) without additional polarization cytokines. Cells were enzymatically lifted with Trypl-E Express Enzyme (1x) (12605028, ThermoFisher) so as not to cleave surface antigen markers. Once lifted, cells were suspended in BV711-CD11B antibody (macrophage marker; BioLegend, 301344, 1:20 dilution) for 30 minutes, to distinguish macrophages from cancer cells in co-culture samples. A macrophage marker for M1 macrophages, anti-human CD80, conjugated to PE-Cyanine7 (305218, BioLegend, 1:20 dilution) and a marker for M2 macrophages, anti-human CD206, conjugated to AlexaFluor 488 (321113, BioLegend, 1:20 dilution) were used to assess polarization status of macrophages at both 24, 48 and 72 hour time points. Macrophages were distinguished from cancer cells based on CD11B positivity, determined by a fluorescent minus one (FMO) CD11B control. FMO controls for macrophage polarization markers were included in these experiments in addition to single color controls for appropriate compensation and gating. CD206+ macrophages were determined based on FMO-CD206 stained M2 activated samples. CD80+ macrophages were similarly determined based on FMO-CD80 stained M1 activated samples. Gating strategy is outlined in Supplemental Figure 2, A-C. Statistical analysis With a minimum expected difference of 30% between conditions, and a standard deviation of 3%, we required at minimum of 3 biological replicates to determine statistical significance at p<0.05. For conditioned media experiments due to a larger standard deviation, we required a minimum of 4 biological replicates. Statistical analyses were done using GraphPad Prism and presented as mean values +/- SEM. Experimental outliers greater than 2 standard deviations from the mean were removed from analysis. Specific tests (2-tailed) and biological replicates are indicated in each figure legend. Biological replicates are indicated as shades of gray on each graph. For all data we assumed gaussian distributions. For flow cytometry data, FlowJo software (version 10.10.0) was used for analysis. For Incucyte assays, the Incucyte Sartorius cell analysis software (version 2021C) was used to measure cell confluence. Image analysis for cell spreading was done using FIJI (version 2.14.0/1.54f). Acknowledgements We would like to acknowledge Keren Hilgendorf, Joseph Casalini, and members of the Roh-Johnson lab for constructive feedback; and James Marvin, Meghan Curtin, and Elena Kurudza for technical support and advice. This work was supported by National Cancer Institute R37CA247994 to MRJ and 5P30CA042014-24 to the Flow Cytometry Core. Footnotes CONFLICT OF INTEREST STATEMENT: The authors declare no potential conflicts of interest. References ↵ Azizi , E. , Carr , A.J. , Plitas , G. , Cornish , A.E. , Konopacki , C. , Prabhakaran , S. , Nainys , J. , Wu , K. , Kiseliovas , V. , Setty , M. , Choi , K. , Fromme , R.M. , Dao , P. , McKenney , P.T. , Wasti , R.C. , Kadaveru , K. , Mazutis , L. , Rudensky , A.Y. , and Pe’er , D . ( 2018 ). Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment . Cell 174 , 1293 – 1308 e1236 . OpenUrl CrossRef PubMed ↵ Boutilier , A.J. , and Elsawa , S.F . ( 2021 ). Macrophage Polarization States in the Tumor Microenvironment . Int J Mol Sci 22 . ↵ Carroll , M.J. , Kapur , A. , Felder , M. , Patankar , M.S. , and Kreeger , P.K . ( 2016 ). M2 macrophages induce ovarian cancer cell proliferation via a heparin binding epidermal growth factor/matrix metalloproteinase 9 intercellular feedback loop . Oncotarget 7 , 86608 – 86620 . OpenUrl ↵ Cendrowicz , E. , Sas , Z. , Bremer , E. , and Rygiel , T.P . ( 2021 ). The Role of Macrophages in Cancer Development and Therapy . Cancers (Basel ) 13 . ↵ Chanput , W. , Mes , J.J. , and Wichers , H.J . ( 2014 ). THP-1 cell line: an in vitro cell model for immune modulation approach . Int Immunopharmacol 23 , 37 – 45 . OpenUrl CrossRef PubMed ↵ Chen , J.J. , Lin , Y.C. , Yao , P.L. , Yuan , A. , Chen , H.Y. , Shun , C.T. , Tsai , M.F. , Chen , C.H. , and Yang , P.C . ( 2005 ). Tumor-associated macrophages: the double-edged sword in cancer progression . J Clin Oncol 23 , 953 – 964 . OpenUrl Abstract / FREE Full Text ↵ Chen , W.X. , Wang , D.D. , Zhu , B. , Zhu , Y.Z. , Zheng , L. , Feng , Z.Q. , and Qin , X.H . ( 2021 ). Exosomal miR-222 from adriamycin-resistant MCF-7 breast cancer cells promote macrophages M2 polarization via PTEN/Akt to induce tumor progression . Aging (Albany NY ) 13 , 10415 – 10430 . OpenUrl ↵ Chen , X. , Yang , M. , Yin , J. , Li , P. , Zeng , S. , Zheng , G. , He , Z. , Liu , H. , Wang , Q. , Zhang , F. , and Chen , D . ( 2022 ). Tumor-associated macrophages promote epithelial-mesenchymal transition and the cancer stem cell properties in triple-negative breast cancer through CCL2/AKT/beta-catenin signaling . Cell Commun Signal 20 , 92 . OpenUrl ↵ Cheng , N. , Bai , X. , Shu , Y. , Ahmad , O. , and Shen , P . ( 2021a ). Targeting tumor-associated macrophages as an antitumor strategy . Biochem Pharmacol 183 , 114354 . OpenUrl ↵ Cheng , S. , Li , Z. , Gao , R. , Xing , B. , Gao , Y. , Yang , Y. , Qin , S. , Zhang , L. , Ouyang , H. , Du , P. , Jiang , L. , Zhang , B. , Yang , Y. , Wang , X. , Ren , X. , Bei , J.X. , Hu , X. , Bu , Z. , Ji , J. , and Zhang , Z . ( 2021b ). A pan-cancer single-cell transcriptional atlas of tumor infiltrating myeloid cells . Cell 184 , 792 – 809 e723 . OpenUrl CrossRef PubMed ↵ Cheng , Y. , Zhong , X. , Nie , X. , Gu , H. , Wu , X. , Li , R. , Wu , Y. , Lv , K. , Leung , G.P. , Fu , C. , Lee , S.M. , Zhang , J. , and Li , J . ( 2023 ). Glycyrrhetinic acid suppresses breast cancer metastasis by inhibiting M2-like macrophage polarization via activating JNK1/2 signaling . Phytomedicine 114 , 154757 . OpenUrl ↵ Cho , H.J. , Jung , J.I. , Lim , D.Y. , Kwon , G.T. , Her , S. , Park , J.H. , and Park , J.H . ( 2012 ). Bone marrow-derived, alternatively activated macrophages enhance solid tumor growth and lung metastasis of mammary carcinoma cells in a Balb/C mouse orthotopic model . Breast Cancer Res 14 , R81 . OpenUrl CrossRef PubMed ↵ Dandekar , R.C. , Kingaonkar , A.V. , and Dhabekar , G.S . ( 2011 ). Role of macrophages in malignancy . Ann Maxillofac Surg 1 , 150 – 154 . OpenUrl ↵ Deng , X.X. , Jiao , Y.N. , Hao , H.F. , Xue , D. , Bai , C.C. , and Han , S.Y . ( 2021 ). Taraxacum mongolicum extract inhibited malignant phenotype of triple-negative breast cancer cells in tumor-associated macrophages microenvironment through suppressing IL-10 / STAT3 / PD-L1 signaling pathways . J Ethnopharmacol 274 , 113978 . OpenUrl ↵ Di Rosa , F. , Cossarizza , A. , and Hayday , A.C. ( 2021 ). To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis . Front Immunol 12 , 653974 . OpenUrl CrossRef ↵ Endl , E. , and Gerdes , J . ( 2000 ). The Ki-67 protein: fascinating forms and an unknown function . Exp Cell Res 257 , 231 – 237 . OpenUrl CrossRef PubMed Web of Science ↵ Engstrom , A. , Erlandsson , A. , Delbro , D. , and Wijkander , J . ( 2014 ). Conditioned media from macrophages of M1, but not M2 phenotype, inhibit the proliferation of the colon cancer cell lines HT-29 and CACO-2 . Int J Oncol 44 , 385 – 392 . OpenUrl PubMed ↵ Eom , D.S. , and Parichy , D.M . ( 2017 ). A macrophage relay for long-distance signaling during postembryonic tissue remodeling . Science 355 , 1317 – 1320 . OpenUrl Abstract / FREE Full Text ↵ Fu , L.Q. , Du , W.L. , Cai , M.H. , Yao , J.Y. , Zhao , Y.Y. , and Mou , X.Z . ( 2020 ). The roles of tumor-associated macrophages in tumor angiogenesis and metastasis . Cell Immunol 353 , 104119 . OpenUrl ↵ Ginhoux , F. , Schultze , J.L. , Murray , P.J. , Ochando , J. , and Biswas , S.K . ( 2016 ). New insights into the multidimensional concept of macrophage ontogeny, activation and function . Nat Immunol 17 , 34 – 40 . OpenUrl CrossRef PubMed ↵ Greiner , D. , Scott , T.M. , Olson , G.S. , Aderem , A. , Roh-Johnson , M. , and Johnson , J.S . ( 2022 ). Genetic Modification of Primary Human Myeloid Cells to Study Cell Migration , Activation, and Organelle Dynamics. Curr Protoc 2 , e514 . OpenUrl CrossRef ↵ Gurevich , D.B. , Severn , C.E. , Twomey , C. , Greenhough , A. , Cash , J. , Toye , A.M. , Mellor , H. , and Martin , P . ( 2018 ). Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression . EMBO J 37 . ↵ Hao , C. , Sheng , Z. , Wang , W. , Feng , R. , Zheng , Y. , Xiao , Q. , and Zhang , B . ( 2023 ). Tumor-derived exosomal miR-148b-3p mediates M2 macrophage polarization via TSC2/mTORC1 to promote breast cancer migration and invasion . Thorac Cancer 14 , 1477 – 1491 . OpenUrl ↵ Harney , A.S. , Arwert , E.N. , Entenberg , D. , Wang , Y. , Guo , P. , Qian , B.Z. , Oktay , M.H. , Pollard , J.W. , Jones , J.G. , and Condeelis , J.S . ( 2015 ). Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA . Cancer Discov 5 , 932 – 943 . OpenUrl Abstract / FREE Full Text ↵ Hinshaw , D.C. , and Shevde , L.A . ( 2019 ). The Tumor Microenvironment Innately Modulates Cancer Progression . Cancer Res 79 , 4557 – 4566 . OpenUrl CrossRef PubMed ↵ Hirano , R. , Okamoto , K. , Shinke , M. , Sato , M. , Watanabe , S. , Watanabe , H. , Kondoh , G. , Kadonosono , T. , and Kizaka-Kondoh , S . ( 2023 ). Tissue-resident macrophages are major tumor-associated macrophage resources, contributing to early TNBC development, recurrence, and metastases . Commun Biol 6 , 144 . OpenUrl ↵ Hughes , R. , Qian , B.Z. , Rowan , C. , Muthana , M. , Keklikoglou , I. , Olson , O.C. , Tazzyman , S. , Danson , S. , Addison , C. , Clemons , M. , Gonzalez-Angulo , A.M. , Joyce , J.A. , De Palma , M. , Pollard , J.W. , and Lewis , C.E. ( 2015 ). Perivascular M2 Macrophages Stimulate Tumor Relapse after Chemotherapy . Cancer Res 75 , 3479 – 3491 . OpenUrl Abstract / FREE Full Text ↵ Italiani , P. , and Boraschi , D . ( 2014 ). From Monocytes to M1/M2 Macrophages: Phenotypical vs. Functional Differentiation . Front Immunol 5 , 514 . OpenUrl CrossRef PubMed ↵ Jamiyan , T. , Kuroda , H. , Yamaguchi , R. , Abe , A. , and Hayashi , M . ( 2020 ). CD68- and CD163-positive tumor-associated macrophages in triple negative cancer of the breast . Virchows Arch 477 , 767 – 775 . OpenUrl CrossRef PubMed ↵ Jedrzejewski , T. , Pawlikowska , M. , Sobocinska , J. , and Wrotek , S . ( 2020 ). Protein-Bound Polysaccharides from Coriolus Versicolor Fungus Disrupt the Crosstalk Between Breast Cancer Cells and Macrophages through Inhibition of Angiogenic Cytokines Production and Shifting Tumour-Associated Macrophages from the M2 to M1 Subtype . Cell Physiol Biochem 54 , 615 – 628 . OpenUrl ↵ Jia , X.H. , Feng , G.W. , Wang , Z.L. , Du , Y. , Shen , C. , Hui , H. , Peng , D. , Li , Z.J. , Kong , D.L. , and Tian , J . ( 2016 ). Activation of mesenchymal stem cells by macrophages promotes tumor progression through immune suppressive effects . Oncotarget 7 , 20934 – 20944 . OpenUrl ↵ Johnson , J.S. , De Veaux , N. , Rives , A.W. , Lahaye , X. , Lucas , S.Y. , Perot , B.P. , Luka , M. , Garcia-Paredes , V. , Amon , L.M. , Watters , A. , Abdessalem , G. , Aderem , A. , Manel , N. , Littman , D.R. , Bonneau , R. , and Menager , M.M. ( 2020 ). A Comprehensive Map of the Monocyte-Derived Dendritic Cell Transcriptional Network Engaged upon Innate Sensing of HIV . Cell Rep 30 , 914 – 931 e919 . OpenUrl CrossRef ↵ Kidwell , C.U. , Casalini , J.R. , Pradeep , S. , Scherer , S.D. , Greiner , D. , Bayik , D. , Watson , D.C. , Olson , G.S. , Lathia , J.D. , Johnson , J.S. , Rutter , J. , Welm , A.L. , Zangle , T.A. , and Roh-Johnson , M . ( 2023 ). Transferred mitochondria accumulate reactive oxygen species, promoting proliferation . Elife 12 . ↵ Kim , K.H. , and Sederstrom , J.M . ( 2015 ). Assaying Cell Cycle Status Using Flow Cytometry . Curr Protoc Mol Biol 111 , 28 26 21 – 28 26 11 . OpenUrl ↵ Kumari , N. , and Choi , S.H . ( 2022 ). Tumor-associated macrophages in cancer: recent advancements in cancer nanoimmunotherapies . J Exp Clin Cancer Res 41 , 68 . OpenUrl CrossRef ↵ Li , D. , Ji , H. , Niu , X. , Yin , L. , Wang , Y. , Gu , Y. , Wang , J. , Zhou , X. , Zhang , H. , and Zhang , Q . ( 2020 ). Tumor-associated macrophages secrete CC-chemokine ligand 2 and induce tamoxifen resistance by activating PI3K/Akt/mTOR in breast cancer . Cancer Sci 111 , 47 – 58 . OpenUrl CrossRef PubMed ↵ Li , J. , Liu , Z. , Wu , X. , Lee , S.M. , Seto , S.W. , Zhang , J. , Zhou , G.C. , and Leung , G.P . ( 2024 ). Anti-metastatic effects of AGS-30 on breast cancer through the inhibition of M2-like macrophage polarization . Biomed Pharmacother 172 , 116269 . OpenUrl ↵ Lin , E.Y. , and Pollard , J.W . ( 2004 ). Macrophages: modulators of breast cancer progression . Novartis Found Symp 256 , 158 – 168 ; discussion 168-172, 259-169. OpenUrl CrossRef PubMed ↵ Lindsten , T. , Hedbrant , A. , Ramberg , A. , Wijkander , J. , Solterbeck , A. , Eriksson , M. , Delbro , D. , and Erlandsson , A . ( 2017 ). Effect of macrophages on breast cancer cell proliferation, and on expression of hormone receptors, uPAR and HER-2 . Int J Oncol 51 , 104 – 114 . OpenUrl ↵ Little , A.C. , Pathanjeli , P. , Wu , Z. , Bao , L. , Goo , L.E. , Yates , J.A. , Oliver , C.R. , Soellner , M.B. , and Merajver , S.D . ( 2019 ). IL-4/IL-13 Stimulated Macrophages Enhance Breast Cancer Invasion Via Rho-GTPase Regulation of Synergistic VEGF/CCL-18 Signaling . Front Oncol 9 , 456 . OpenUrl ↵ Liu , C. , Wu , C. , Yang , Q. , Gao , J. , Li , L. , Yang , D. , and Luo , L . ( 2016 ). Macrophages Mediate the Repair of Brain Vascular Rupture through Direct Physical Adhesion and Mechanical Traction . Immunity 44 , 1162 – 1176 . OpenUrl CrossRef PubMed ↵ Liu , J. , Geng , X. , Hou , J. , and Wu , G . ( 2021 ). New insights into M1/M2 macrophages: key modulators in cancer progression . Cancer Cell Int 21 , 389 . OpenUrl CrossRef PubMed ↵ Liu , S.Q. , Zhou , Z.Y. , Dong , X. , Guo , L. , and Zhang , K.J . ( 2020 ). LncRNA GNAS-AS1 facilitates ER+ breast cancer cells progression by promoting M2 macrophage polarization via regulating miR-433-3p/GATA3 axis . Biosci Rep 40 . ↵ Locati , M. , Curtale , G. , and Mantovani , A . ( 2020 ). Diversity, Mechanisms, and Significance of Macrophage Plasticity . Annu Rev Pathol 15 , 123 – 147 . OpenUrl PubMed ↵ Ma , C. , He , D. , Tian , P. , Wang , Y. , He , Y. , Wu , Q. , Jia , Z. , Zhang , X. , Zhang , P. , Ying , H. , Jin , Z.B. , and Hu , G . ( 2022 ). miR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression . Proc Natl Acad Sci U S A 119 . ↵ Mahmoud , S.M. , Lee , A.H. , Paish , E.C. , Macmillan , R.D. , Ellis , I.O. , and Green , A.R . ( 2012 ). Tumour-infiltrating macrophages and clinical outcome in breast cancer . J Clin Pathol 65 , 159 – 163 . OpenUrl Abstract / FREE Full Text ↵ Mantovani , A. , Marchesi , F. , Malesci , A. , Laghi , L. , and Allavena , P . ( 2017 ). Tumour-associated macrophages as treatment targets in oncology . Nat Rev Clin Oncol 14 , 399 – 416 . OpenUrl CrossRef PubMed ↵ Mantovani , A. , Sozzani , S. , Locati , M. , Allavena , P. , and Sica , A . ( 2002 ). Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes . Trends Immunol 23 , 549 – 555 . OpenUrl CrossRef PubMed Web of Science ↵ Martinez , F.O. , and Gordon , S . ( 2014 ). The M1 and M2 paradigm of macrophage activation: time for reassessment . F1000Prime Rep 6 , 13 . OpenUrl ↵ Mohd Yasin , Z.N. , Mohd Idrus , F.N. , Hoe , C.H. , and Yvonne-Tee , G.B. ( 2022 ). Macrophage polarization in THP-1 cell line and primary monocytes: A systematic review . Differentiation 128 , 67 – 82 . OpenUrl CrossRef ↵ Mor , G. , Yue , W. , Santen , R.J. , Gutierrez , L. , Eliza , M. , Berstein , L.M. , Harada , N. , Wang , J. , Lysiak , J. , Diano , S. , and Naftolin , F . ( 1998 ). Macrophages, estrogen and the microenvironment of breast cancer . J Steroid Biochem Mol Biol 67 , 403 – 411 . OpenUrl CrossRef PubMed Web of Science ↵ Moradi-Chaleshtori , M. , Bandehpour , M. , Heidari , N. , Mohammadi-Yeganeh , S. , and Mahmoud Hashemi , S . ( 2021 ). Exosome-mediated miR-33 transfer induces M1 polarization in mouse macrophages and exerts antitumor effect in 4T1 breast cancer cell line . Int Immunopharmacol 90 , 107198 . OpenUrl ↵ Moraes , L.A. , Kar , S. , Foo , S.L. , Gu , T. , Toh , Y.Q. , Ampomah , P.B. , Sachaphibulkij , K. , Yap , G. , Zharkova , O. , Lukman , H.M. , Fairhurst , A.M. , Kumar , A.P. , and Lim , L.H.K . ( 2017 ). Annexin-A1 enhances breast cancer growth and migration by promoting alternative macrophage polarization in the tumour microenvironment . Sci Rep 7 , 17925 . OpenUrl ↵ Mosser , D.M. , and Edwards , J.P . ( 2008 ). Exploring the full spectrum of macrophage activation . Nat Rev Immunol 8 , 958 – 969 . OpenUrl CrossRef PubMed Web of Science ↵ Mu , X. , Shi , W. , Xu , Y. , Xu , C. , Zhao , T. , Geng , B. , Yang , J. , Pan , J. , Hu , S. , Zhang , C. , Zhang , J. , Wang , C. , Shen , J. , Che , Y. , Liu , Z. , Lv , Y. , Wen , H. , and You , Q . ( 2018 ). Tumor-derived lactate induces M2 macrophage polarization via the activation of the ERK/STAT3 signaling pathway in breast cancer . Cell Cycle 17 , 428 – 438 . OpenUrl CrossRef ↵ Muller , S. , Kohanbash , G. , Liu , S.J. , Alvarado , B. , Carrera , D. , Bhaduri , A. , Watchmaker , P.B. , Yagnik , G. , Di Lullo , E. , Malatesta , M. , Amankulor , N.M. , Kriegstein , A.R. , Lim , D.A. , Aghi , M. , Okada , H. , and Diaz , A. ( 2017 ). Single-cell profiling of human gliomas reveals macrophage ontogeny as a basis for regional differences in macrophage activation in the tumor microenvironment . Genome Biol 18 , 234 . OpenUrl CrossRef PubMed ↵ Munir , M.T. , Kay , M.K. , Kang , M.H. , Rahman , M.M. , Al-Harrasi , A. , Choudhury , M. , Moustaid-Moussa , N. , Hussain , F. , and Rahman , S.M . ( 2021 ). Tumor-Associated Macrophages as Multifaceted Regulators of Breast Tumor Growth . Int J Mol Sci 22 . ↵ Najafi , M. , Hashemi Goradel , N. , Farhood , B. , Salehi , E. , Nashtaei , M.S. , Khanlarkhani , N. , Khezri , Z. , Majidpoor , J. , Abouzaripour , M. , Habibi , M. , Kashani , I.R. , and Mortezaee , K . ( 2019 ). Macrophage polarity in cancer: A review . J Cell Biochem 120 , 2756 – 2765 . OpenUrl CrossRef PubMed ↵ Nascimento , C.R. , Rodrigues Fernandes , N.A. , Gonzalez Maldonado , L.A. , and Rossa Junior , C . ( 2022 ). Comparison of monocytic cell lines U937 and THP-1 as macrophage models for in vitro studies . Biochem Biophys Rep 32 , 101383 . OpenUrl ↵ Nie , X. , Fu , L. , Cheng , Y. , Wu , X. , Lv , K. , Li , R. , Wu , Y. , Leung , G.P. , Fu , C. , Lee , S.M. , Seto , S.W. , Zhang , J. , and Li , J . ( 2023 ). Garcinone E suppresses breast cancer growth and metastasis by modulating tumor-associated macrophages polarization via STAT6 signaling . Phytother Res 37 , 4442 – 4456 . OpenUrl ↵ Nielsen , S.R. , and Schmid , M.C . ( 2017 ). Macrophages as Key Drivers of Cancer Progression and Metastasis . Mediators Inflamm 2017 , 9624760 . OpenUrl CrossRef ↵ Obeid , E. , Nanda , R. , Fu , Y.X. , and Olopade , O.I . ( 2013 ). The role of tumor-associated macrophages in breast cancer progression (review) . Int J Oncol 43 , 5 – 12 . OpenUrl PubMed ↵ Pan , Y. , Yu , Y. , Wang , X. , and Zhang , T . ( 2020 ). Tumor-Associated Macrophages in Tumor Immunity . Front Immunol 11 , 583084 . OpenUrl PubMed ↵ Ramesh , A. , Brouillard , A. , Kumar , S. , Nandi , D. , and Kulkarni , A . ( 2020 ). Dual inhibition of CSF1R and MAPK pathways using supramolecular nanoparticles enhances macrophage immunotherapy . Biomaterials 227 , 119559 . OpenUrl ↵ Riabov , V. , Gudima , A. , Wang , N. , Mickley , A. , Orekhov , A. , and Kzhyshkowska , J . ( 2014 ). Role of tumor associated macrophages in tumor angiogenesis and lymphangiogenesis . Front Physiol 5 , 75 . OpenUrl CrossRef PubMed ↵ Roh-Johnson , M. , Bravo-Cordero , J.J. , Patsialou , A. , Sharma , V.P. , Guo , P. , Liu , H. , Hodgson , L. , and Condeelis , J . ( 2014 ). Macrophage contact induces RhoA GTPase signaling to trigger tumor cell intravasation . Oncogene 33 , 4203 – 4212 . OpenUrl CrossRef PubMed Web of Science ↵ Shapouri-Moghaddam , A. , Mohammadian , S. , Vazini , H. , Taghadosi , M. , Esmaeili , S.A. , Mardani , F. , Seifi , B. , Mohammadi , A. , Afshari , J.T. , and Sahebkar , A . ( 2018 ). Macrophage plasticity, polarization, and function in health and disease . J Cell Physiol 233 , 6425 – 6440 . OpenUrl CrossRef PubMed ↵ Shrivastava , R. , Asif , M. , Singh , V. , Dubey , P. , Ahmad Malik , S. , Lone , M.U. , Tewari , B.N. , Baghel , K.S. , Pal , S. , Nagar , G.K. , Chattopadhyay , N. , and Bhadauria , S . ( 2019 ). M2 polarization of macrophages by Oncostatin M in hypoxic tumor microenvironment is mediated by mTORC2 and promotes tumor growth and metastasis . Cytokine 118 , 130 – 143 . OpenUrl ↵ Sica , A. , and Mantovani , A . ( 2012 ). Macrophage plasticity and polarization: in vivo veritas . J Clin Invest 122 , 787 – 795 . OpenUrl CrossRef PubMed Web of Science ↵ Sousa , S. , Brion , R. , Lintunen , M. , Kronqvist , P. , Sandholm , J. , Monkkonen , J. , Kellokumpu-Lehtinen , P.L. , Lauttia , S. , Tynninen , O. , Joensuu , H. , Heymann , D. , and Maatta , J.A . ( 2015 ). Human breast cancer cells educate macrophages toward the M2 activation status . Breast Cancer Res 17 , 101 . OpenUrl ↵ Stachowicz-Suhs , M. , Labedz , N. , Anisiewicz , A. , Banach , J. , Klopotowska , D. , Milczarek , M. , Piotrowska , A. , Dziegiel , P. , Maciejczyk , A. , Matkowski , R. , and Wietrzyk , J . ( 2024 ). Calcitriol promotes M2 polarization of tumor-associated macrophages in 4T1 mouse mammary gland cancer via the induction of proinflammatory cytokines . Sci Rep 14 , 3778 . OpenUrl ↵ Steenbrugge , J. , Breyne , K. , Demeyere , K. , De Wever , O. , Sanders , N.N. , Van Den Broeck , W. , Colpaert , C. , Vermeulen , P. , Van Laere , S. , and Meyer , E. ( 2018 ). Anti-inflammatory signaling by mammary tumor cells mediates prometastatic macrophage polarization in an innovative intraductal mouse model for triple-negative breast cancer . J Exp Clin Cancer Res 37 , 191 . OpenUrl CrossRef ↵ Taciak , B. , Bialasek , M. , Braniewska , A. , Sas , Z. , Sawicka , P. , Kiraga , L. , Rygiel , T. , and Krol , M . ( 2018 ). Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages . PLoS One 13 , e0198943 . OpenUrl CrossRef ↵ Tang , C. , Jiang , S.T. , Li , C.X. , Jia , X.F. , and Yang , W.L . ( 2024 ). The Effect of Salvianolic Acid A on Tumor-Associated Macrophage Polarization and Its Mechanisms in the Tumor Microenvironment of Triple-Negative Breast Cancer . Molecules 29 . ↵ Tao , S. , Chen , Q. , Lin , C. , and Dong , H . ( 2020 ). Linc00514 promotes breast cancer metastasis and M2 polarization of tumor-associated macrophages via Jagged1-mediated notch signaling pathway . J Exp Clin Cancer Res 39 , 191 . OpenUrl ↵ van Engeland , M. , Nieland , L.J. , Ramaekers , F.C. , Schutte , B. , and Reutelingsperger , C.P. ( 1998 ). Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure . Cytometry 31 , 1 – 9 . OpenUrl CrossRef PubMed Web of Science ↵ Xiang , X. , Wang , J. , Lu , D. , and Xu , X . ( 2021 ). Targeting tumor-associated macrophages to synergize tumor immunotherapy . Signal Transduct Target Ther 6 , 75 . OpenUrl ↵ Yu , Y. , Hao , J. , Wang , L. , Zheng , X. , Xie , C. , Liu , H. , Wu , J. , Qiao , S. , and Shi , J . ( 2023 ). Astragaloside IV antagonizes the malignant progression of breast cancer induced by macrophage M2 polarization through the TGF-beta-regulated Akt/Foxo1 pathway . Pathol Res Pract 249 , 154766 . OpenUrl ↵ Yuan , J. , He , H. , Chen , C. , Wu , J. , Rao , J. , and Yan , H . ( 2019 ). Combined high expression of CD47 and CD68 is a novel prognostic factor for breast cancer patients . Cancer Cell Int 19 , 238 . OpenUrl ↵ Yunna , C. , Mengru , H. , Lei , W. , and Weidong , C. ( 2020 ). Macrophage M1/M2 polarization . Eur J Pharmacol 877 , 173090 . OpenUrl PubMed ↵ Zhang , C. , Wei , S. , Dai , S. , Li , X. , Wang , H. , Zhang , H. , Sun , G. , Shan , B. , and Zhao , L . ( 2023a ). The NR_109/FUBP1/c-Myc axis regulates TAM polarization and remodels the tumor microenvironment to promote cancer development . J Immunother Cancer 11 . ↵ Zhang , H. , Liu , L. , Liu , J. , Dang , P. , Hu , S. , Yuan , W. , Sun , Z. , Liu , Y. , and Wang , C . ( 2023b ). Roles of tumor-associated macrophages in anti-PD-1/PD-L1 immunotherapy for solid cancers . Mol Cancer 22 , 58 . OpenUrl ↵ Zhang , L. , Nie , F. , Zhao , J. , Li , S. , Liu , W. , Guo , H. , and Yang , P . ( 2024 ). PGRN is involved in macrophage M2 polarization regulation through TNFR2 in periodontitis . J Transl Med 22 , 407 . OpenUrl ↵ Zhang , M. , He , Y. , Sun , X. , Li , Q. , Wang , W. , Zhao , A. , and Di , W. ( 2014 ). A high M1/M2 ratio of tumor-associated macrophages is associated with extended survival in ovarian cancer patients . J Ovarian Res 7 , 19 . OpenUrl CrossRef PubMed ↵ Zhang , Q. , and Sioud , M . ( 2023 ). Tumor-Associated Macrophage Subsets: Shaping Polarization and Targeting . Int J Mol Sci 24 . ↵ Zhang , Y. , Fan , Y. , Jing , X. , Zhao , L. , Liu , T. , Wang , L. , Zhang , L. , Gu , S. , Zhao , X. , and Teng , Y . ( 2021 ). OTUD5-mediated deubiquitination of YAP in macrophage promotes M2 phenotype polarization and favors triple-negative breast cancer progression . Cancer Lett 504 , 104 – 115 . OpenUrl ↵ Zhao , L. , Wang , G. , Qi , H. , Yu , L. , Yin , H. , Sun , R. , Wang , H. , Zhu , X. , and Yang , A. ( 2024 ). LINC00330/CCL2 axis-mediated ESCC TAM reprogramming affects tumor progression . Cell Mol Biol Lett 29 , 77 . OpenUrl ↵ Zhao , Y. , Yu , Z. , Ma , R. , Zhang , Y. , Zhao , L. , Yan , Y. , Lv , X. , Zhang , L. , Su , P. , Bi , J. , Xu , H. , He , M. , and Wei , M . ( 2021 ). lncRNA-Xist/miR-101-3p/KLF6/C/EBPalpha axis promotes TAM polarization to regulate cancer cell proliferation and migration . Mol Ther Nucleic Acids 23 , 536 – 551 . OpenUrl ↵ Zheng , P. , Luo , Q. , Wang , W. , Li , J. , Wang , T. , Wang , P. , Chen , L. , Zhang , P. , Chen , H. , Liu , Y. , Dong , P. , Xie , G. , Ma , Y. , Jiang , L. , Yuan , X. , and Shen , L . ( 2018 ). Tumor-associated macrophages-derived exosomes promote the migration of gastric cancer cells by transfer of functional Apolipoprotein E . Cell Death Dis 9 , 434 . OpenUrl CrossRef PubMed ↵ Zhu , S. , Luo , Z. , Li , X. , Han , X. , Shi , S. , and Zhang , T . ( 2021 ). Tumor-associated macrophages: role in tumorigenesis and immunotherapy implications . J Cancer 12 , 54 – 64 . OpenUrl CrossRef PubMed ↵ Zhukova , O.V. , Kovaleva , T.F. , Arkhipova , E.V. , Ryabov , S.A. , and Mukhina , I.V . ( 2020 ). Tumor-associated macrophages: Role in the pathological process of tumorigenesis and prospective therapeutic use (Review) . Biomed Rep 13 , 47 . OpenUrl Back to top Previous Next Posted June 01, 2024. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Macrophage subtypes inhibit breast cancer proliferation in culture Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Macrophage subtypes inhibit breast cancer proliferation in culture Sophia R.S. Varady , Daniel Greiner , Minna Roh-Johnson bioRxiv 2024.06.01.596963; doi: https://doi.org/10.1101/2024.06.01.596963 Share This Article: Copy Citation Tools Macrophage subtypes inhibit breast cancer proliferation in culture Sophia R.S. Varady , Daniel Greiner , Minna Roh-Johnson bioRxiv 2024.06.01.596963; doi: https://doi.org/10.1101/2024.06.01.596963 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7917) Biochemistry (18516) Bioengineering (14666) Bioinformatics (43839) Biophysics (22308) Cancer Biology (19412) Cell Biology (26582) Clinical Trials (138) Developmental Biology (13825) Ecology (20730) Epidemiology (2067) Evolutionary Biology (25183) Genetics (16031) Genomics (23285) Immunology (18460) Microbiology (41984) Molecular Biology (17831) Neuroscience (92269) Paleontology (689) Pathology (2947) Pharmacology and Toxicology (5032) Physiology (8008) Plant Biology (15783) Scientific Communication and Education (2087) Synthetic Biology (4506) Systems Biology (10128) Zoology (2360) window.__CF$cv$params={r:'a2fa23884ca3c165',t:'MTc4NzQ4ODMzNA==',u:'01a02e9b792571d3a8396fd6dce5a721',ut:'MwmBb2ABWhjruvGQQDPNbL8e1ysgcjfEhjmLZtUd3lY-1787488336-1.2.1.1-RaesffagELfVQprlfh5ocF1JvnKZZs8fqGMVwg.edM9WkzJHUOfddPbKER1dRrgNW2o_bBMJytP9pzWBH8mlY2H2vFHRYSYjo_.5fThjDTk',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();