Abstract
Simple SummaryTrastuzumab and radiation are used clinically to treat HER2-overexpressing breast cancers; however, the mechanistic synergy of anti-HER2 and radiation therapy has not been investigated. In this study, we identify that a subtherapeutic dose of trastuzumab sensitizes the tumor microenvironment to fractionated radiation. This results in longitudinal sustained response by triggering a state of innate immune activation through reduced DNA damage repair and increased tumor oxygenation. As positron emission tomography imaging can be used to longitudinally evaluate changes in tumor hypoxia, synergy of combination therapies is the result of both cellular and molecular changes in the tumor microenvironment.DNA damage repair and tumor hypoxia contribute to intratumoral cellular and molecular heterogeneity and affect radiation response. The goal of this study is to investigate anti-HER2-induced radiosensitization of the tumor microenvironment to enhance fractionated radiotherapy in models of HER2+ breast cancer. This is monitored through in vitro and in vivo studies of phosphorylated γ-H2AX, [18F]-fluoromisonidazole (FMISO)-PET, and transcriptomic analysis. In vitro, HER2+ breast cancer cell lines were treated with trastuzumab prior to radiation and DNA double-strand breaks (DSB) were quantified. In vivo, HER2+ human cell line or patient-derived xenograft models were treated with trastuzumab, fractionated radiation, or a combination and monitored longitudinally with [18F]-FMISO-PET. In vitro DSB analysis revealed that trastuzumab administered prior to fractionated radiation increased DSB. In vivo, trastuzumab prior to fractionated radiation significantly reduced hypoxia, as detected through decreased [18F]-FMISO SUV, synergistically improving long-term tumor response. Significant changes in IL-2, IFN-gamma, and THBS-4 were observed in combination-treated tumors. Trastuzumab prior to fractionated radiation synergistically increases radiotherapy in vitro and in vivo in HER2+ breast cancer which is independent of anti-HER2 response alone. Modulation of the tumor microenvironment, through increased tumor oxygenation and decreased DNA damage response, can be translated to other cancers with first-line radiation therapy.
Highlights
Tumor hypoxia is an integral component of the tumor microenvironment that impacts tumor aggressiveness, metastatic potential, and response to therapy [1]
BT474 cells exhibited 50.3 ± 9% cell death on day 7, while BT474 cells treated with trastuzumab prior to fractionated radiation 7e6x±hi5b.i3te%dc7e6ll ±de5a.3th%(pce=ll0.d0e1a).thW(hpe=n 0tr.e0a1t)e.dWwhiethn stirnegaltee-ddowseitrhadsiinatgiloen,dBoTse47r4adceialltsioexnh, iBbTit4e7d4 7c6e.3lls±ex6h.1i%bitceedll7d6e.a3t±h6o.n1%dacyel7l,dweahtihleoBnTd4a7y4 c7e, lwlshtirleeaBteTd4w74itcheltlrsatsrteuaztuemd awbitphritorarsttousziunmglaebdporsieoratdoisaitniognlee-xdhoisbeitreadd6ia9t.i3o±n e2x.1h%ibicteeldl d6e9a.3th±(2p.1=%0.c1e3l)l(dFeigauthre(p1B=)0. .13) (Figure 1B)
When treated with single-dose radiation, SKBR3 cells exhibited 54 ± 2.6% cell dieteadth6, 2w.3h±ile1.S5K%BcRe3llcdeellastthre(pat=ed0.w01i)t.hWtrhasetnutzruemataebd pwriitohr stoinsgilneg-dleo-dseosreadraiadtiiaotnio, nSKexBhRi3bicteeldls 6e2x.3hi±bit2e.d1%54c±el2l.6d%eactehll(pde=at0h.0, w1)h(iFleigSuKreBR1D3 c).elWlshtreenatteredawteidthwtriathstufrzaucmtioanbapterdiorratodisaitnigolne,MdDosAe -rMadBi-a4t5io3nceexllhsibexithedibi6t2e.d3 1±92.7.1%± c0e.6ll%decaetlhl d(pea=th0,.0w1)h(iFleigMurDe A1D-M).BW-4h5e3ncterlelsatterdeawteidth wfritahcttiroansatutezdumraadbiaptiroionr, tMoDfrAac-tMioBn-a4t5e3dcrealdlsiaetxiohnibeixtehdib1it9e.7d ±610.±6%6.1ce%llcdeelladthe,atwhh(iple=M0.D01A).When treated with single-dose radiation, MDA-MB-453 cells exhibited 59 ± 2.6% cell death, while MDA-MB-453 cells treated with trastuzumab prior to single-dose radiation exhibited 57.7 ± 0.6% cell death (p = 0.44) (Figure 1E)
Summary
Tumor hypoxia is an integral component of the tumor microenvironment that impacts tumor aggressiveness, metastatic potential, and response to therapy [1]. Radiation therapy converts oxygen present in tissue into reactive oxygen species (ROS), which will damage the DNA and cause genomic instability in cancerous tissue [1,3]. HER2 expression has been found to have a negative effect on radiation response, highlighting the need to optimize anti-HER2 targeted therapies with clinically relevant radiation therapy [7,8]. Potent longitudinal radiosensitizers must improve ROS production and accommodate oxygenation-induced changes in DNA damage response. Liu et al notes that hypoxic conditions trigger E2F4 binding, which mechanistically decreases Rad non-homologous end joining, suggesting that an increase in tumor oxygenation may improve tumor DNA damage response [11]. An effective radiosensitizer must induce ROS production, while accounting for the increased DNA damage, highlighting the need for a multi-faceted cellular and molecular-based radiosensitizer
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