2.1.

Cells and Reagents

A murine medullary mammary adenocarcinoma syngeneic with C57Bl/6 mice (E0771, Roswell Park Cancer Institute, Buffalo, NY) was maintained in RPMI 1640 medium (Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% gamma-irradiated fetal calf serum (HyClone/Thermo-Fisher, Waltham, MA) and Primocin (InvivoGen, San Diego, CA). Cells were passaged no more than five times before being replaced from frozen stocks. After harvesting with 25% trypsin/ethylenediaminetetraacetic acid (EDTA), cells were centrifuged and resuspended in sterile phosphate buffered saline (PBS) and kept on ice until implantation into a mammary fat pad. T47D and MCF-7 human breast cancer cell lines [American Type Culture Collection (ATCC), Manassas, VA] were also cultured in RPMI 1640 medium for use in in vitro proliferation assays. RAW264.7 transformed murine macrophages (ATCC) were used as a positive control for TNF-$\alpha$ production and were cultured in Dulbecco’s modified Eagle's medium (DMEM) supplemented with $4.5\mathrm{g}/\mathrm{L}$ glucose, 10% fetal calf serum (FCS), and Primocin. To activate RAW264.7 cells, lipopolysaccharide (LPS, Escherichia coli serotype 026:B6, Sigma-Aldrich, St. Louis, MO) was added to media at $100\mathrm{ng}/\mathrm{mL}$ for 24 h prior to assaying for TNF-$\alpha$. All lines were tested for mycoplasma contamination bi-monthly using MycoFluor detection kit (Invitrogen, Carlsbad, CA) and only certified mycoplasma-free cultures were used for implantation.

2.2.

Purification of Tumor CD11b+ Cells by Magnetic Separation

E0771 tumors grown in mammary fat pads were finely minced, washed with RPMI 1640 and then shaken in medium supplemented with $0.5\mathrm{mg}/\mathrm{mL}$ of collagenase type D (Sigma-Aldrich, St. Louis, MO) at 37°C for 180 min. The cell dispersion was passed through metal mesh (100-μm pore size) and washed three times in RPMI 1640, then resuspended in MACS buffer (Miltenyi Biotec, Auburn, CA) containing 2% EDTA and 5% bovine serum albumin (BSA) in PBS. To harvest ${\mathrm{CD}11\mathrm{b}}^{+}$ cells, the single-cell suspension was incubated with anti-CD11b antibody conjugated to magnetic beads (Miltenyi Biotec, Auburn, CA). The cell suspension was then applied to a type LS positive selection column with MidiMACS (Miltenyi Biotec) according to manufacturer instructions. Selected cells were cultured to $\sim 70\%$ confluence in DMEM supplemented with 10% FCS and Primocin.

2.3.

Animals and Husbandry

C57Bl/6 female mice (Jackson Laboratories, Bar Harbor, ME) were used between 15 and 19 weeks of age. Mice were housed in two-way static (nonventilated) conditions in groups of five, and were allowed ad libitum access to standard food and water. To determine the effects of global deletion of TNF-$\alpha$, female B6.129S-${\mathrm{Tnf}}^{tm1Gkl}/\mathrm{J}$ (Jackson Labs) mice were used between 15 and 19 weeks of age and housed as above. C57Bl/6 animals represent a valid control genotype for this knockout. All animal work was done in accordance with University Committee for Animal Resources regulations.

2.4.

Tumor Implantation and Liposome Administration

Animals were anesthetized with a ketamine/xylazine mixture ($90/9\mathrm{mg}/\mathrm{kg}$ body weight) delivered intraperitoneally (i.p.). The ventral surface of the animal was depilated and $1\times {10}^5$ E0771 cells were implanted in the right inguinal mammary fat pad using a 27-gauge needle. Four hours following this procedure, mice were administered either PBS- or clodronate-containing liposomes (Encapsula NanoSciences, Nashville, TN, $5\mathrm{mg}/\mathrm{mL}$ stock solution) i.p. at $0.4\mathrm{mL}/20\mathrm{g}$ body weight on day 0.²⁹ After the first injection, liposomes were injected every third day at $0.2\mathrm{mL}/20\mathrm{g}$ body weight. The long and short axes of each tumor were measured on days 3, 9, 15, 21, and 27 with digital calipers. Tumor volume was calculated using the formula for a prolate spheroid $[\mathrm{V}=(4/3)\pi N^{2}L]$ and standardized to volume at day 3 (multiplicity). On day 27 postimplantation, animals were sacrificed by sodium pentobarbital overdose and subsequent cervical dislocation. Half the tumor was frozen in dry ice for sectioning and immunohistochemistry. The other half was placed in cell culture media and prepared for flow cytometric analysis. Lungs were placed in 10% neutral-buffered formalin for hematoxylin and eosin (H&E) staining.

2.5.

Flow Cytometry

To prepare a single cell suspension, tumors were finely minced, washed with RPMI 1640, and strained through a 70-μm mesh filter to remove debris. After centrifuging two times, the cells were counted by cytometer with trypan blue exclusion. Viable cells ($2\times {10}^6$) were incubated at 4°C for 15 min in ACK buffer to lyse red blood cells, centrifuged, and resuspended in flow wash buffer consisting of 1% BSA and 25% sodium azide in sterile PBS. Cells were incubated for 30 min with 25-μL anti-mouse CD16/CD32 antibody at $1:50$ dilution (BD Pharmingen, Franklin Lakes, NJ) to block $F_c\gamma$ receptors and then were resuspended in 100 μL buffer containing either: (1) buffer only (autofluorescence); (2) $1:50$ peridinin chlorophyll-conjugated rat ${\mathrm{IgG}}_{2a}$ [BD Pharmingen (isotype control)], or (3) fluorescein isothiocyanate (FITC)-conjugated rat (${\mathrm{IgG}}_{2a}$) anti-mouse F4/80 (Abcam, Cambridge, MA). Cells were incubated in the dark for 30 min at 4°C. Cells were then centrifuged in flow wash buffer twice and resuspended in 4% paraformaldehyde. Samples were stored in the dark at 4°C until cytometric analysis using a FACSAria flow cytometer (BD Biosciences, Franklin Lakes, NJ) equipped with FlowDiVa software.

2.6.

TNF-$\alpha$ Determination

A Quantikine enzyme-linked immunosorbent assay (ELISA) kit specific for mouse TNF-$\alpha$ (R&D Systems, Minneapolis) with a sensitivity of $5.1\mathrm{pg}/\mathrm{mL}$ was employed to measure TNF-$\alpha$ in cell culture supernatants and tumor lysates. To prepare cell culture supernatants, cells were grown to 70% confluence in a T-75 tissue culture flask and then media was removed and replaced with reduced serum media for 48 h. This media was removed, centrifuged, and immediately assayed for TNF-$\alpha$. Tumor lysates were prepared by homogenization of excised tumor tissue in RIPA buffer containing sodium deoxycholate and HALT protease/phosphatase inhibitor (Pierce Protein Research, Rockford, IL). Lysates were centrifuged at $\mathrm{18,000}\times \mathrm{g}$ for 10 min and supernatants were removed for assay. Total protein concentration in tumor lysates was determined with a bicinchoninic acid assay (Pierce Protein Research, Rockford, IL) per manufacturer specifications. For the ELISA, absorption was measured at 450 nm using a mutliwell plate reader (Synergy HT, BioTek Instruments Inc, Winooski, VT). Curve fitting and sample concentration calculations were conducted with Gen5 software (Biotek). Results are presented as TNF-$\alpha$ concentration standardized to sample protein concentration.

2.7.

Proliferation Assays

In vitro proliferation was determined with the use of a fluorescent CyQuant Cell Proliferation Assay (Molecular Probes/Invitrogen, Carlsbad, CA) following manufacturer’s instructions. To detect DNA fluorescence, a plate reader (BioTek) exciting bound dye at 480 nm and detecting at 520 nm was used. This assay effectively measures viable cell number, and we have determined that fluorescent intensity correlates directly with actual cell number (data not shown).

2.8.

Immunohistochemistry

Snap-frozen tumor halves were sectioned at 7 μm on a cryostat (Reichert-Jung, Depew, NY) at $-21°\mathrm{C}$ and static-mounted on positively charged slides (VWR, Radnor, Pennsylvania). Each slide contained at least four sections from each experimental group to minimize possible staining artifacts between samples. Sections were fixed in a $3:1$ mixture of acetone/methanol for 20 min at $-20°\mathrm{C}$. Slides were rehydrated twice in sterile PBS for 5 min and then placed in peroxidase blocking solution (5% BSA, 2% Triton X-100) for 1 h, followed by two 5 min PBS washes. Sections were incubated at room temperature for 2 h in a humidified chamber with PBS containing 5% BSA and combinations of the following antibodies: (1) rabbit anti-mouse collagen I (Abcam, Cambridge, MA), $1:200$ dilution; (2) FITC-conjugated rat ${\mathrm{IgG}}_{2a}$ anti-mouse CD31 (BD Pharmingen, Franklin Lakes, NJ), 1:500 dilution; or (3) FITC-conjugated rat ${\mathrm{IgG}}_{2a}$ anti-mouse F4/80, $1:50$ dilution. To detect rabbit anti-mouse collagen I, sections were then washed $2\times 5\min$ and incubated for 2 h at room temperature in AlexaFluor 594-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA), $1:500$ dilution. Optimal antibody dilutions were predetermined. Slides were then washed and coverslipped in Prolong Gold AntiFade without DAPI (Invitrogen, Carlsbad, CA) and allowed to dry 24 h before imaging.

2.9.

Evaluation of Metastatic Burden

Automated H&E staining (Dako, Carpinteria, CA) was performed on 3 μm rotary microtome sections of paraffin-embedded lungs. H&E-stained lung sections were obtained at 50 μm intervals through both lobes of the lung and evaluated by a blinded observer using brightfield microscopy (Olympus BX-51, Center Valley, PA). Malignant cells lose their differentiation and undergo increased proliferation, frequently exhibiting enlarged and variable sized nuclei as well as evidence of accelerated mitosis. Consequently, accepted criteria for recognizing metastatic foci in H&E-stained sections include a high ratio of hematoxylin relative to eosin, surrounding abnormalities in lung structure, abnormal shape/size of nuclei and/or presence of abnormal mitotic spindles, and differences in cell shape and size.³⁰ We used these criteria to identify lung metastases, with results presented as the number of metastases in 10 sections per animal.

2.10.

Imaging and Image Analysis

Slides were imaged by a blinded observer using a custom-built two-photon microscope (Olympus). Two-photon (2P) excitation was achieved by a MaiTai Ti:sapphire laser providing 100 fs pulses at 80 MHz and 810 nm. Beam scanning and image acquisition were performed with a Fluoview FV300 scanning system interfaced with a BX61WI upright microscope (Olympus, Center Valley, PA). The focusing objective was a XLUMPLFL20xW water immersion lens ($20\times$, 0.95 N.A., Olympus). The objective focused the 810 nm excitation beam on the sample and at the same time collected both backscattered SHG signal and the two-photon excited IF from the antibody of interest. The backscattered SHG and IF signals were separated from the excitation beam by a short pass dichroic mirror (670 DCSX, Chroma, Rockingham, VT), with the SHG and IF emissions then separated into two channels with a long pass dichroic mirror (475 DCSX, Chroma, Rockingham, VT). SHG was collected with a bandpass filter centered at 405 nm (HQ405/30m-2P, Chroma) and detected by a photomultiplier tube (PMT) (HC125-02 Hamamatsu Corporation, Hamamatsu, Japan). The IF signal was collected with a bandpass filter centered at 635 nm (HQ635/30m-2P, Chroma). For capturing this red IF channel, a red-sensitive PMT was used (Hamamatsu HC125-01), and the emission signal was additionally filtered (Chroma E700SP-2P) to block any stray near-infrared infra-red (IR) laser light. Resulting images are 680 μm across. Laser power was monitored and kept constant throughout the experiment and across experimental repetitions, as were PMT voltage, gain, and offset. Ten tissue slices were chosen by a blinded observer, spaced throughout each tumor half (the other half is used to verify macrophage depletion) and five fields of view were generated from each slice. The five imaged fields of view formed an “x” pattern; one in the tumor center, then four adjacent diagonal fields of view (i.e., one to the upper left of the first central image, with the corners of the images not quite touching, one to the upper right of the first central image, etc.). Hence, 50 image pairs were analyzed as described below to produce a single average SHG/IF value for each tumor.

Image analysis was performed as follows. In IMAGE J (Rasband, W. S., IMAGE J, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2011), the background in each channel (SHG and IF) was defined by the average pixel counts of an image with no excitation laser and subtracted from the raw SHG and IF images, respectively. A single common threshold was then chosen by a blinded observer which passed the most collagen pixels while rejecting the most background pixels (typically individual pixels not clearly laying in a fibrillar pattern). The common threshold was then applied to all SHG images, setting collagen fiber pixels to 1 and any remaining dim background pixels to zero, producing an “SHG mask.” The same thresholding procedure was followed with IF images, producing an “IF mask.” The SHG image was multiplied by the SHG mask, producing the “masked SHG image,” and the IF image was multiplied by the IF mask to produce the “masked IF image.” The average pixel count of the masked SHG image, divided by the average pixel count of the SHG mask, is then the average pixel count of those pixels above threshold, i.e., within collagen fibers, likewise with the masked IF image. The average SHG pixel count, average IF pixel count, and their ratio, of those pixels within collagen fibers, are then reported as the images’ SHG, IF, and OI, respectively. Note that due to fluctuations in pixel counts, there are occasional pixels which are above threshold in the SHG image but below threshold (and hence set to zero) in the IF image. Consequently, the SHG/IF ratio is infinity for those pixels. This makes it problematic to calculate an average SHG/IF for the imaged region by first producing an SHG/IF ratio image and then calculating the average pixel count of that image. We avoid this problem by first separately calculating the average SHG and IF pixel counts of the whole imaged region and then dividing these two (nonzero) numbers to produce the SHG/IF for the whole imaged region.

2.11.

Statistical Analysis

Statistical analysis was performed using Prism 5 software (GraphPad, La Jolla, CA). Student’s (unpaired) $t$-tests were employed to make pairwise comparisons where appropriate. To analyze the effects of macrophage depletion and TNF-$\alpha$ knockout, two-way analysis of variance (ANOVA) was used. Significant main effects or interactions were analyzed with Bonferroni posttests to adjust for multiple comparisons. Tumor growth curves were assessed by two-way ANOVA with repeated measures, followed by multiple group comparisons using Bonferroni posthoc analysis. Probability values ($p$) less than or equal to 0.05 were considered significant differences between groups. When $N$ is reported for animal experiments in the figures or text, it represents the number of animals studied.

3.1.

E0771 does not Produce TNF-$\alpha$ nor Proliferate in Response to TNF-$\alpha$ In Vitro or In Vivo

Pilot experiments were conducted to determine: (1) if E0771 cells produce TNF-$\alpha$ and (2) if E0771 responds to TNF-$\alpha$. E0771 cells cultured with 10% FCS to 75% confluence did not produce measurable TNF-$\alpha$ [$<5.1\mathrm{pg}/\mathrm{mL}$, Fig. 1(a)]. By comparison, RAW 264.7 macrophages were also tested at 75% confluence in both normal and LPS-activated conditions and produced significant levels of TNF-$\alpha$ with or without activation by LPS [Fig. 1(a)]. To confirm that TAMs from E0771 tumors produce TNF-$\alpha$, macrophages were isolated from E0771 tumors using magnetic antibody separation targeted to CD11b.³¹ These TAMs also produced TNF-$\alpha$ at detectable levels in culture [Fig. 1(a)]. Furthermore, E0771 tumors were grown in both wild-type and TNF-${\alpha }^{(-/-)}$ animals. TNF-$\alpha$ was readily detectable in wild-type-derived E0771 tumor lysates at $1:1$, $1:10$, and $1:25$ dilutions, but not in those grown in TNF-${\alpha }^{(-/-)}$ mice, where levels were again undetectable [$<5.1\mathrm{pg}/\mathrm{mL}$, Fig. 1(b)]. Taken together, these results suggest that the E0771 cell line is not a source of TNF-$\alpha$ production in vitro or in vivo and that E0771-associated macrophages, as expected, are a primary source of this signal in vivo.

Fig. 1

E0771 breast cancer cells do not produce nor respond to TNF-$\alpha$. (a): In vitro TNF-$\alpha$ production: Supernatants from E0771 tumor cells showed no detectable TNF-$\alpha$ in vitro (E0771 bar). Supernatants from $\mathrm{CD}11\mathrm{b}+$ TAMs isolated from E0771 tumors, in contrast, did produce detectable TNF-$\alpha$ ($\mathrm{E}0771/\mathrm{CD}11\mathrm{b}+$ bar). To confirm sensitivity of the assay, RAW264.7-transformed murine macrophages produced detectable TNF-$\alpha$ both when unstimulated (RAW264.7 bar) and upon activation with $100\mathrm{ng}/\mathrm{mL}$ LPS for 24 h (RAW264.7+LPS bar). TNF-$\alpha$ ELISA sensitivity was $5.1\mathrm{pg}/\mathrm{mL}$, $n=8$ samples for all but $\mathrm{E}0771/\mathrm{CD}11{\mathrm{b}}^{+}$, where $n=5$. Both the media alone control (media bar) and E0771 supernatants (E0771 bar) registered below sensitivity (not detectable). (b): In vivo TNF-$\alpha$ production: also by this same ELISA assay, TNF-$\alpha$ was detectable in E0771 tumors grown in C57Bl/6 mice (wt bar) but not in mice lacking TNF-$\alpha$ [TNF-${\alpha }^{(-/-)}$ bar]. (c): Proliferation responses to TNF-$\alpha$: three breast tumor cell lines (T47D, MCF-7, and E0771) were treated with media control or $20\mathrm{ng}/\mathrm{mL}$ TNF-$\alpha$. Pairwise comparisons indicate that T47D proliferation was significantly elevated by $20\mathrm{ng}/\mathrm{mL}$ TNF-$\alpha$ at 48 h ($p<0.01$), MCF-7 proliferation was significantly decreased ($p<0.05$), whereas only E0771 proliferation was unchanged in response to TNF-$\alpha$. Proliferation was assessed by fluorescent intensity of CyQuant DNA-binding dye standardized to that of cells in untreated media, $n=10$ per group.

The proliferative response of E0771 to TNF-$\alpha$ was determined using a fluorescent DNA-binding dye kit (CyQuant). In another model of breast cancer (T47D), TNF-$\alpha$ was shown to directly promote mitogenic signaling by binding to TNFR1 and activating p42/p44 MAPK, JNK, PI3-K/Akt pathways, and NF-$\kappa B$ transcriptional activation.³² Furthermore, in that model, application of TNF-$\alpha$ supported tumor growth. Conversely, TNF-$\alpha$ induced apoptosis in MCF-7 breast cancer cells in vitro.³³ To determine the response of E0771 cells to TNF-$\alpha$, we incubated cells for 48 h in either media or media containing $20\mathrm{ng}/\mathrm{mL}$ TNF-$\alpha$ [Fig. 1(c)]. TNF-$\alpha$ did not significantly alter proliferation of E0771, in direct contrast to the human breast tumor cell line T47D, which showed a marked increase in proliferation in response to $20\mathrm{ng}/\mathrm{mL}$ TNF-$\alpha$, as reported.³² Furthermore, MCF-7 cells exposed to this level of TNF-$\alpha$ exhibited significant reduction in proliferation, also as expected.³³ Therefore, TNF-$\alpha$ does not significantly stimulate E0771 proliferation nor significantly inhibit its growth.

3.2.

ClodL Selectively Impairs Macrophage Viability In Vitro and Depletes TAMs In Vivo

ClodL are commonly used to selectively deplete macrophages in tumors.²⁹ Macrophages that phagocytose ClodL are destroyed by release of clodronate into the cytoplasm following degradation of the liposome shell and subsequent formation of a lethal metabolite.³⁴ To determine if TAM susceptibility to ClodL is specific, we tested the viability of E0771 cells, fibroblasts (HFF-1, transformed human foreskin fibroblasts), and RAW264.7, a transformed murine macrophage cell line in the presence of PBS-containing liposomes (PBSL) or ClodL. ClodL did not alter viability of E0771 or HFF-1 cells relative to PBSL-treated controls. By contrast, RAW264.7 viability was significantly reduced by ClodL [Fig. 2(a)]. Depletion of TAMs in vivo was quantified by flow cytometric analysis of F4/80, a marker for mature macrophages.³⁵ ClodL treatment in E0771-bearing mice dramatically reduced the percentage of F4/80-positive cells in E0771 tumors in both wild-type and TNF-${\alpha }^{(-/-)}$ mice [Fig. 2(b)]. Together, these results demonstrate that ClodL treatment is selective for TAMs in E0771 tumors. Figure 2(b) also demonstrates that the percentage of E0771-associated F4/80-positive macrophages is not statistically different between wild-type and TNF-${\alpha }^{(-/-)}$ mice ($p=0.35$).

Fig. 2

ClodL selectively reduces macrophage viability in vitro and in vivo. (a) Graded dose–response treatments with Clodronate (0 to $0.1\mathrm{mg}/\mathrm{mL}$) or PBS control liposomes demonstrated that at all Clodronate concentrations tested, only macrophage RAW264.7 cells, but not nonmacrophage E0771 breast tumor cells or HFF-1 fibroblasts, were sensitive to Clodronate depletion ($n=8$ each data point, $p<0.05$ for RAW264.7 PBSL versus ClodL at all doses). For each cell type, fluorescent intensities were standardized to the PBS control liposome group (no ClodL), to account for differences in proliferative rate inherent to cell type. Liposomes containing PBS (vehicle) only also did not affect proliferation of RAW264.7 macrophages at any liposome concentration (RAW264.7/PBSL trace). (b) Flow cytometric analysis indicated that endogenous $\mathrm{F}4/{80}^{+}$ TAMs were depleted by in vivo Clodronate treatment in both wild-type (wt) and TNF-${\alpha }^{(-/-)}$ mice [$n=21\mathrm{wt}/\mathrm{PBSL}$; $n=16\mathrm{wt}/\mathrm{ClodL}$; $n=15$ TNF-${\alpha }^{(-/-)}/\mathrm{PBSL}$; $n=15$ TNF-${\alpha }^{(-/-)}/\mathrm{ClodL}$]. Moreover, these data also indicate that under baseline (PBSL, i.e., no depletion) conditions, $\mathrm{F}4/{80}^{+}$ macrophage populations were not significantly different in wt versus TNF-${\alpha }^{(-/-)}$) animals.

3.3.

Effects of TAM Depletion and TNF-$\alpha$ Knockout on Intensity of SHG-Producing Fibers

To determine if TAM depletion or TNF-$\alpha$ knockout affects the intensity of SHG-producing fibers in the E0771 tumor, tumors were grown orthotopically in either wild-type or TNF-${\alpha }^{(-/-)}$ mice treated with liposomes containing clodronate to deplete TAMs. Control mice were treated with liposomes containing PBS. SHG imaging of excised E0771 tumors reveals a typical fibrillar structure (Fig. 3). Figure 4(a) shows that either TAM depletion or TNF-$\alpha$ knockout reduced the intensity of SHG-producing fibers in E0771 tumors. By two-way ANOVA, a strong main effect of TAM depletion ($p<0.0001$) and a strong interaction between TAM depletion and TNF-$\alpha$ knockout ($p<0.0004$) was revealed. By Bonferroni’s posttest for multiple comparisons, the intensity of SHG-producing fibers in wild-type/PBSL mice was significantly elevated relative to all other groups ($p<0.05$). Intensity of SHG-producing fibers in tumors from TNF-${\alpha }^{(-/-)}/\mathrm{PBSL}$ mice was also greater than that in tumors from wild-type/ClodL mice ($p<0.05$) but not relative to TNF-${\alpha }^{(-/-)}/\mathrm{ClodL}$ mice ($p=0.31$). Intensity of SHG-producing fibers did not differ between the two ClodL-treated groups. These results demonstrate that TAM depletion or attenuation of stromal TNF-$\alpha$ reduces intensity of SHG-producing fibers in the E0771 tumor. Furthermore, in the absence of TAMs, the inability to produce TNF-$\alpha$ did not produce a greater effect than either treatment alone. This suggests that nonmacrophage sources of TNF-$\alpha$ do not significantly influence SHG intensity.

Fig. 3

SHG image of E0771 murine mammary adenocarcinoma excised from the mammary fat pad of a C57Bl/6 mouse. The tumor sample was excised, sectioned, and fixed. With 810-nm excitation, typical collagen fibrils are imaged via SHG at 405-nm emission.

Fig. 4

Effects of macrophage depletion and TNF-$\alpha$ knockout on SHG and IF signals in the E0771 tumor. (a) SHG-producing fibers in E0771 tumors in wild-type/PBSL mice were significantly brighter compared to all other groups ($p<0.05$ for all comparisons). In addition, SHG-producing fibers in E0771 tumors in wild-type/ClodL mice were significantly dimmer compared to TNF-${\alpha }^{(-/-)}/\mathrm{PBSL}$ tumors ($p<0.05$). No other group was found significantly different from another ($p>0.05$). $n=21\mathrm{wt}/\mathrm{PBSL}$, $16\mathrm{wt}/\mathrm{ClodL}$, $n=15$TNF-${\alpha }^{(-/-)}/\mathrm{PBSL}$, and $n=15$ TNF-${\alpha }^{(-/-)}/\mathrm{ClodL}$). (b) Collagen I immunofluorescence (IF) was significantly lower in E0771 tumors in wild-type/PBSL mice compared to all other groups ($p<0.05$ for all comparisons). In addition, collagen I IF in E0771 tumors in TNF-${\alpha }^{(-/-)}/\mathrm{PBSL}$ mice was significantly lower than in E0771 tumors in TNF-${\alpha }^{(-/-)}/\mathrm{ClodL}$ mice. No other group was found significantly different from another ($p>0.05$).

3.4.

Effects of TAM Depletion and TNF-$\alpha$ Knockout on Collagen Fiber IF Intensity

SHG is dependent upon collagen content as well as fiber microstructure. Therefore, in order to produce a more direct measure of fiber microstructure, we normalize the SHG intensity to the total amount of collagen present. As collagen type I is the primary fibrillar collagen in vivo,^20,21 we quantify collagen fibril content using fluorescently labeled anticollagen type I antibodies.^16,17 TAM depletion and stromal TNF-$\alpha$ knockout both alter detected collagen I fiber IF in the E0771 tumor [Fig. 4(b)]. By two-way ANOVA, we see a strong main effect of both TAM depletion and TNF-$\alpha$ knockout (both $p<0.0001$) and a significant degree of interaction ($p=0.04$) was detected. By multiple comparison analysis, anticollagen type I IF was significantly lower in wild-type/PBSL mice compared to the three other groups ($p<0.05$). Furthermore, although IF signal in tumors grown in wild-type/ClodL mice is statistically equivalent to that of both TNF-$\alpha$ knockout groups ($p>0.05$), tumors grown in TNF-${\alpha }^{(-/-)}/\mathrm{PBSL}$ mice exhibit less IF than do tumors grown in TNF-${\alpha }^{(-/-)}/\mathrm{ClodL}$ mice ($p<0.05$). We therefore conclude that tumor growth in the presence of TAM depletion results in a significant increase in anticollagen type I IF in E0771 tumors. Growth during attenuation of stromal TNF-$\alpha$ accomplishes this to the same extent. Furthermore, in the absence of TAMs, depletion of stromal TNF-$\alpha$ has no further effect on IF. However, in the absence of stromal TNF-$\alpha$, depletion of TAMs further increases IF. The combined abrogation of TAMs and TNF-$\alpha$ did not produce a greater increase in IF signal intensity than TAM depletion alone.

3.5.

Effects of TAM Depletion and TNF-$\alpha$ Knockout on OI

Division of the SHG signal by the IF signal produces the order index (OI), which is primarily sensitive to changes in fibrillar microstructure, such as fibril diameter, spacing, and order versus disorder in fibrillar packing.^11–15 In Fig. 5, OI was reduced by TAM depletion with ClodL or stromal knockout of TNF-$\alpha$, with a main effect of $p<0.0001$ and $p=0.013$, respectively, with a significant interaction ($p=0.0027$). Bonferroni post-ho$c$ analysis shows that wild-type/PBSL mice have significantly elevated OI relative to the three remaining groups ($p<0.05$). However, none of the three remaining groups exhibit significant differences between their mean OI values ($p>0.05$). We therefore conclude that tumor growth in the presence of TAM depletion results in a significant decrease in OI in E0771 tumors, as does growth during attenuation of stromal TNF-$\alpha$, to the same extent. Furthermore, combined abrogation of TAMs and TNF-$\alpha$ did not produce a greater decrease in OI than either depletion alone.

Fig. 5

Effects of macrophage depletion and TNF-$\alpha$ knockout on OI. E0771 tumors grown in wild-type/PBSL mice had a significantly higher order index (OI, defined as SHG intensity/IF intensity) relative to all other treatment groups ($p<0.05$). No other group was found significantly different from another ($p>0.05$).

3.6.

Metastatic Burden Correlates Negatively with Both TAM Presence and TNF-$\alpha$ Expression

To evaluate the possibility of a relationship between modulations in OI and modulations in metastatic burden, lungs were resected, sectioned, and stained with H&amp;E and then imaged to determine the extent of metastasis (Fig. 6). E0771 readily metastasizes to the lung from the primary site,³⁶ and two-way ANOVA shows a strong main effect of both TAM depletion and abrogation of TNF-$\alpha$ on metastatic burden ($p=0.0006$ and $p<0.0001$, respectively). The degree of interaction is again significant ($p=0.0007$). Bonferroni posthoc analysis shows that while wild-type/PBSL mice exhibit significantly ($p<0.05$) higher metastatic burden than any of the other groups, the other three groups do not show distinguishably different metastatic burdens ($p>0.05$). We conclude that both TAM depletion and TNF-$\alpha$ knockout result in significant decreases in metastatic events and that these decreases correlate well with OI values in the E0771 tumor.

Fig. 6

Lung metastatic burden decreases sharply with both macrophage depletion and TNF-$\alpha$ knockout. E0771 tumors grown in wild-type/PBSL mice generated significantly higher numbers of lung metastases, compared to E0771 tumors in TAM depleted and TNF-$\alpha$ knockout mice ($p<0.05$). Metastatic output was assessed across 10 sections of lung per group. No other group was found significantly different from another ($p>0.05$). $n=21\mathrm{wt}/\mathrm{PBSL}$, $n=16\mathrm{wt}/\mathrm{ClodL}$, $n=15$TNF-${\alpha }^{(-/-)}/\mathrm{PBSL}$, and $n=15$ TNF-${\alpha }^{(-/-)}/\mathrm{ClodL}$.

3.7.

ClodL and TNF-$\alpha$ Knockout Reduce E0771 Tumor Growth In Vivo

To determine if TAM depletion or TNF-$\alpha$ knockout affect tumor growth, E0771 was implanted in PBSL- or ClodL-treated wild-type or TNF-${\alpha }^{(-/-)}$ mice and tumor volume was measured over the course of 27 days. The data presented in Fig. 7 were normalized at each time point relative to the volume at day 3 posttumor inoculation. By repeated measure of two-way ANOVA, a treatment by time interaction was revealed ($p<0.0005$). Wild-type/PBSL mice bore significantly larger tumors at day 27 ($p<0.05$, Bonferroni posttest) compared to any other group. Tumor growth did not differ among the other three groups.

Fig. 7

E0771 tumor volume multiplicity decreases with TAM depletion or TNF-$\alpha$ knockout, but the effects are not synergistic. After 27 days, E0771 tumors grown in wild-type mice on PBSL therapy ($n=21$) were larger than those grown in wild-type mice on ClodL therapy ($n=16$), as well as those grown in TNF-$\alpha$ knockout mice exposed to either PBSL or ClodL ($p<0.05$, $n=15$ for both). No other group was found significantly different from another at 27 days, and no significant differences in tumor volume multiplicity were found at earlier timepoints ($p>0.05$). Tumor growth was assessed by normalizing measurements to tumor size at day 3 (i.e., tumor volume multiplicity) to minimize the possibility that slightly different numbers of cells may have been injected despite constant injection volumes and cell densities.