Introduction_NN1 
It_PNP has_VHZ been_VBN postulated_VVN that_CJT the_AT0 functional_AJ0 plasticity_NN1 of_PRF the_AT0 adult_NN1 mammary_AJ0 epithelium_NN1 ,_PUN which_DTQ is_VBZ required_VVN for_PRP response_NN1 to_PRP hormonal_AJ0 changes_NN2 during_PRP pregnancy_NN1 and_CJC lactation_NN1 ,_PUN may_VM0 make_VVI the_AT0 breast_NN1 tissue_NN1 more_AV0 susceptible_AJ0 to_PRP replaced-dns_NN2 mechanisms_NN2 of_PRF normal_AJ0 mammary_AJ0 epithelial_AJ0 cell_NN1 (_PUL MEC_NP0 )_PUR biology_NN1 that_CJT contribute_VVB to_PRP tumorigenesis_NN1 is_VBZ critically_AV0 important_AJ0 for_PRP furthering_VVG our_DPS understanding_NN1 of_PRF breast_NN1 cancer_NN1 and_CJC extending_VVG the_AT0 survival_NN1 of_PRF breast_NN1 cancer_NN1 patients_NN2 ._SENT Of_PRF particular_AJ0 interest_NN1 are_VBB postpartum_NN1 breast_NN1 cancers_NN2 (_PUL PPBCs_NP0 )_PUR ,_PUN defined_VVN as_PRP breast_NN1 cancers_NN2 diagnosed_VVN in_PRP women_NN2 within_PRP 10_CRD years_NN2 of_PRF most_DT0 recent_AJ0 childbirth_NN1 ,_PUN as_CJS these_DT0 cancers_NN2 are_VBB nearly_AV0 three_CRD times_NN2 more_AV0 likely_AJ0 to_TO0 result_VVI in_PRP lethal_AJ0 metastasis_NN1 ._SENT 
The_AT0 majority_NN1 of_PRF mammary_AJ0 morphogenesis_NN1 occurs_VVZ postnatally_AV0 beginning_VVG at_PRP puberty_NN1 and_CJC culminating_VVG with_PRP lactation_NN1 following_AJ0 pregnancy_NN1 ._SENT As_CJS such_DT0 ,_PUN differentiation_NN1 of_PRF the_AT0 mammary_AJ0 epithelium_NN1 is_VBZ not_XX0 complete_AJ0 until_PRP a_AT0 full_AJ0 pregnancy-lactation_NN1 cycle_NN1 ._SENT Upon_PRP weaning_VVG ,_PUN or_CJC after_PRP pregnancy_NN1 in_PRP the_AT0 absence_NN1 of_PRF nursing_NN1 ,_PUN the_AT0 mammary_AJ0 gland_NN1 regresses_VVZ to_TO0 resemble_VVI the_AT0 pre-pregnant_AJ0 architecture_NN1 through_PRP a_AT0 process_NN1 termed_VVN postpartum_NN1 involution_NN1 ._SENT In_PRP mice_NN2 ,_PUN postpartum_NN1 involution_NN1 occurs_VVZ in_PRP two_CRD primary_AJ0 phases_NN2 ._SENT In_PRP the_AT0 first_ORD phase_NN1 ,_PUN milk_NN1 proteins_NN2 accumulate_VVB in_PRP the_AT0 mammary_AJ0 ducts_NN2 and_CJC alveoli_NN2 resulting_VVG in_PRP downstream_AJ0 phosphorylation_NN1 and_CJC activation_NN1 of_PRF signal_NN1 transducer_NN1 and_CJC activator_NN1 of_PRF transcription_NN1 (_PUL pSTAT3_CRD )_PUR ,_PUN and_CJC subsequent_AJ0 lysosomal-mediated_AJ0 cell_NN1 death_NN1 ._SENT In_PRP mice_NN2 ,_PUN if_CJS pups_NN2 are_VBB returned_VVN within_PRP 48_CRD h_ZZ0 of_PRF weaning_VVG ,_PUN this_DT0 process_NN1 can_VM0 be_VBI reversed_VVN ,_PUN and_CJC lactation_NN1 can_VM0 continue_VVI ._SENT If_CJS pups_NN2 are_VBB not_XX0 returned_VVN ,_PUN however_AV0 ,_PUN the_AT0 irreversible_AJ0 second_ORD phase_NN1 of_PRF involution_NN1 begins_VVZ ._SENT This_DT0 phase_NN1 is_VBZ characterized_VVN by_PRP degradation_NN1 of_PRF the_AT0 mammary_AJ0 basement_NN1 membrane_NN1 by_PRP matrix_NN1 metalloproteinases_NN2 and_CJC anoikis-mediated_AJ0 cell_NN1 death_NN1 in_PRP the_AT0 absence_NN1 of_PRF laminin-mediated_AJ0 activation_NN1 of_PRF integrin_NN1 alpha-6_CRD /_PUN beta-1_CRD (_PUL α6β1_CRD )_PUR and_CJC downstream_AJ0 pro-survival_NN1 signaling_VVG ._SENT 
SEMA7A_NN1 ,_PUN or_CJC CD108w_NP0 ,_PUN is_VBZ a_AT0 member_NN1 of_PRF the_AT0 semaphorin_AJ0 protein_NN1 family_NN1 ,_PUN named_VVN after_PRP the_AT0 English_AJ0 verb_NN1 "_PUQ semaphore_NN1 "_PUQ ,_PUN which_DTQ means_VVZ to_TO0 transmit_VVI a_AT0 signal_NN1 across_PRP a_AT0 long_AJ0 distance_NN1 ._SENT SEMA7A_NP0 was_VBD first_ORD identified_VVN for_PRP its_DPS expression_NN1 on_PRP lymphocytes_NN2 ,_PUN but_CJC has_VHZ since_AV0 been_VBN shown_VVN to_TO0 exert_VVI diverse_AJ0 roles_NN2 in_PRP neuronal_AJ0 development_NN1 ,_PUN cell_NN1 differentiation_NN1 ,_PUN ECM_NP0 deposition_NN1 ,_PUN various_AJ0 components_NN2 of_PRF the_AT0 immune_AJ0 system_NN1 ,_PUN and_CJC pathologies_NN2 including_PRP fibrosis_NN1 and_CJC cancer_NN1 ._SENT Though_CJS elevated_AJ0 SEMA7A_NN1 is_VBZ observed_VVN in_PRP breast_NN1 cancer_NN1 ,_PUN where_CJS we_PNP have_VHB established_VVN a_AT0 role_NN1 for_PRP it_PNP in_PRP conferring_VVG cell_NN1 survival_NN1 ,_PUN in_PRP part_NN1 ,_PUN via_PRP promotion_NN1 of_PRF anoikis_NN1 resistance_NN1 ,_PUN the_AT0 role_NN1 of_PRF SEMA7A_NN1 in_PRP normal_AJ0 MECs_NN2 has_VHZ not_XX0 previously_AV0 been_VBN investigated_VVN ._SENT SEMA7A_NN1 is_VBZ known_VVN to_TO0 signal_VVI through_PRP β1-integrin_NN1 in_PRP immune_AJ0 cells_NN2 ,_PUN neurons_NN2 ,_PUN and_CJC cancer_NN1 ;_PUN β1-integrin_NP0 also_AV0 mediates_VVZ MEC_NN1 survival_NN1 during_PRP development_NN1 ,_PUN in_PRP part_NN1 ,_PUN via_PRP phosphorylation_NN1 and_CJC activation_NN1 of_PRF downstream_AJ0 pro-survival_NN1 signaling_VVG mediated_VVN by_PRP PI3-kinase_NN1 and_CJC protein_NN1 kinase_NN1 B_ZZ0 ,_PUN or_CJC Akt1_NP0 ._SENT Though_CJS it_PNP is_VBZ known_VVN that_CJT redundant_AJ0 secretory_AJ0 epithelial_AJ0 cells_NN2 die_VVB during_PRP postpartum_NN1 involution_NN1 ,_PUN the_AT0 factor_NN1 (_PUL s_ZZ0 )_PUR that_CJT allow_VVB some_DT0 cells_NN2 to_TO0 survive_VVI and_CJC regenerate_VVI a_AT0 lactation-competent_AJ0 gland_NN1 for_PRP future_AJ0 pregnancies_NN2 remain_VVB unknown_AJ0 ._SENT Herein_AV0 ,_PUN we_PNP have_VHB identified_VVN enrichment_NN1 for_PRP SEMA7A_NP0 expressing_VVG α6β1_CRD positive_AJ0 MECs_NN2 during_PRP the_AT0 matrix_NN1 remodeling_AJ0 period_NN1 of_PRF postpartum_NN1 involution_NN1 and_CJC a_AT0 role_NN1 for_PRP SEMA7A_NN1 in_PRP specifically_AV0 promoting_VVG the_AT0 survival_NN1 of_PRF α6-integrin_NP0 +_NP0 luminal_NN1 progenitor_NN1 cells_NN2 ._SENT We_PNP provide_VVB evidence_NN1 that_CJT exogenous_AJ0 stimulation_NN1 of_PRF immortalized_VVN MECs_NN2 with_PRP purified_AJ0 SEMA7A_NN1 protein_NN1 promotes_VVZ anoikis_NN1 resistance_NN1 via_PRP activation_NN1 of_PRF α6_NN1 and_CJC β1-integrins_NN2 and_CJC activation_NN1 of_PRF phosphorylated_AJ0 AKT_NN1 (_PUL pAKT_NP0 )_PUR ._SENT Consistent_AJ0 with_PRP a_AT0 role_NN1 for_PRP SEMA7A_NN1 in_PRP promoting_VVG anoikis_NN1 resistance_NN1 ,_PUN SEMA7A_NN1 knockout_NN1 mice_NN2 (_PUL SEMA7Atmalk1_CRD /_PUN J_ZZ0 )_PUR exhibit_VVB phenotypes_NN2 associated_VVN with_PRP accelerated_AJ0 involution_NN1 including_PRP early_AJ0 presence_NN1 of_PRF apoptotic_AJ0 cell_NN1 bodies_NN2 in_PRP mammary_AJ0 lumens_NN2 ,_PUN increased_AJ0 pSTAT3_NN1 ,_PUN and_CJC premature_AJ0 reappearance_NN1 of_PRF adipocytes_NN2 ._SENT α6-integrin_NP0 also_AV0 drives_VVZ maintenance_NN1 of_PRF tissue_NN1 resident_NN1 stem_NN1 cells_NN2 and_CJC therapy_NN1 resistant_AJ0 cancer_NN1 stem_NN1 cells_NN2 and_CJC our_DPS findings_NN2 reveal_VVB that_CJT SEMA7A_NN1 drives_VVZ stem_NN1 cell_NN1 phenotypes_NN2 and_CJC chemoresistance_NN1 in_PRP cultured_AJ0 MECs_NN2 in_PRP a_AT0 manner_NN1 that_DT0 is_VBZ dependent_AJ0 on_PRP α6_CRD and_CJC β1_CRD ._SENT Finally_AV0 ,_PUN we_PNP provide_VVB evidence_NN1 for_PRP SEMA7A_NN1 in_PRP driving_VVG transformed_AJ0 phenotypes_NN2 in_PRP normal_AJ0 MECs_NN2 and_CJC reversing_VVG transformed_AJ0 phenotypes_NN2 in_PRP genetically_AV0 identical_AJ0 ,_PUN ras-transformed_AJ0 ,_PUN variants_NN2 of_PRF the_AT0 same_DT0 cell_NN1 line_NN1 ._SENT Our_DPS results_NN2 are_VBB the_AT0 first_ORD to_TO0 identify_VVI a_AT0 role_NN1 for_PRP SEMA7A_NN1 in_PRP anoikis_NN1 resistance_NN1 and_CJC promotion_NN1 of_PRF stem_NN1 and_CJC cancer_NN1 cell_NN1 phenotypes_NN2 via_PRP activation_NN1 of_PRF α6_NN1 and_CJC β1-integrins_NN2 ._SENT 
Methods_NN2 
Cell_NN1 culture_NN1 
MCF10A_NP0 /_PUN MCF12A_NP0 cells_NN2 were_VBD obtained_VVN from_PRP ,_PUN and_CJC cultured_AJ0 ,_PUN according_VVG to_PRP ATCC_NP0 ._SENT replaced-dns_NP0 cells_NN2 were_VBD obtained_VVN from_PRP K_NP0 ._SENT Polyak_NP0 (_PUL University_NN1 of_PRF Harvard_NP0 ,_PUN Cambridge_NP0 ,_PUN MA_NP0 )_PUR ._SENT Cells_NN2 were_VBD validated_VVN by_PRP the_AT0 DNA_NN1 sequencing_NN1 core_NN1 at_PRP the_AT0 University_NN1 of_PRF Colorado_NP0 Anschutz_NP0 Medical_AJ0 Campus_NN1 and_CJC found_VVD to_TO0 be_VBI pure_AJ0 populations_NN2 of_PRF their_DPS respective_AJ0 cell_NN1 lines_NN2 ._SENT Cells_NN2 were_VBD regularly_AV0 tested_VVN for_PRP mycoplasma_NN1 throughout_PRP the_AT0 studies_NN2 ._SENT Cells_NN2 were_VBD sub-cultured_AJ0 as_CJS previously_AV0 described_VVN (_PUL reference_NN1 )_PUR ,_PUN or_CJC according_VVG to_PRP ATCC_NP0 standards_NN2 ._SENT Exogenous_AJ0 SEMA7A_NN1 protein_NN1 utilized_VVN in_PRP our_DPS studies_NN2 was_VBD purified_VVN from_PRP media_NN0 of_PRF cells_NN2 stably_AV0 transfected_VVN with_PRP an_AT0 Fc-tagged_AJ0 version_NN1 of_PRF SEMA7A_NN1 by_PRP published_AJ0 methods_NN2 ._SENT Control_NN1 plasmid_NN1 (_PUL pcDNA3.1_CRD )_PUR was_VBD obtained_VVN from_PRP H_NP0 ._SENT Ford_NP0 (_PUL University_NN1 of_PRF Colorado_NP0 Anschutz_NP0 Medical_AJ0 Campus_NN1 ,_PUN Denver_NP0 ,_PUN CO_NN1 )_PUR ._SENT 
shRNA_NN1 knockdown_NN1 and_CJC overexpression_NN1 
shRNA_NN1 plasmids_NN2 targeting_VVG Sema7a_NN1 ,_PUN and_CJC a_AT0 negative_AJ0 control_NN1 shRNA_NN1 (_PUL SABiosciences_NP0 )_PUR ,_PUN were_VBD amplified_VVN in_PRP E_NP0 ._SENT coli_NN2 and_CJC plasmid_NN1 DNA_NN1 was_VBD isolated_VVN by_PRP Plasmid_NN1 Maxi-Prep_NP0 (_PUL Qiagen_NP0 )_PUR ._SENT replaced-dns_NN2 cells_NN2 were_VBD cultured_VVN overnight_AV0 to_PRP ~_NN1 80_CRD %_NN0 confluence_NN1 ._SENT 1_CRD μg_NN1 /_PUN μL_NN1 of_PRF each_DT0 shRNA_NN1 was_VBD added_VVN to_PRP Transfectagro_NP0 Reduced-Serum_NN1 Medium_NN1 (_PUL Corning_NP0 )_PUR and_CJC incubated_VVN for_PRP 15_CRD min_NN0 with_PRP 4_CRD μL_NN1 of_PRF X-treme_NP0 Gene_NP0 HP_NN1 DNA_NN1 transfection_NN1 reagent_NN1 (_PUL Roche_NP0 )_PUR ._SENT Transfected_AJ0 cells_NN2 were_VBD selected_VVN for_PRP hygromycin_NN1 resistance_NN1 ._SENT Stable_AJ0 knockdown_NN1 was_VBD confirmed_VVN by_PRP qPCR_NN1 as_CJS shown_VVN in_PRP Black_NP0 et_NP0 al_NP0 ._SENT Negative_AJ0 control_NN1 cells_NN2 were_VBD transfected_VVN with_PRP a_AT0 scrambled_AJ0 artificial_AJ0 sequence_NN1 not_XX0 matching_VVG human_AJ0 ,_PUN mouse_NN1 ,_PUN or_CJC rat_NN1 ._SENT Overexpression_NN1 plasmid_NN1 (_PUL SEMA7A-Fc_NP0 )_PUR was_VBD a_AT0 generous_AJ0 gift_NN1 from_PRP R_NP0 ._SENT Medzhitov_NP0 (_PUL Yale_NP0 University_NN1 ,_PUN New_NP0 Haven_NP0 ,_PUN CT_NP0 )_PUR ._SENT All_DT0 other_AJ0 over-expression_NN1 plasmids_NN2 (_PUL p304-V5-Blasticidin_NP0 and_CJC V5-SEMA7A_NP0 )_PUR were_VBD obtained_VVN from_PRP the_AT0 Functional_AJ0 Genomics_NN1 Core_NN1 at_PRP the_AT0 CUAnschutz_NP0 Medical_AJ0 Campus_NN1 and_CJC overexpression_NN1 was_VBD confirmed_VVN via_PRP qPCR_NN1 and_CJC western_AJ0 blot_NN1 analysis_NN1 ._SENT KD_NP0 and_CJC overexpression_NN1 as_CJS previously_AV0 described_VVN ._SENT 
Animal_NN1 studies_NN2 
All_DT0 animal_NN1 procedures_NN2 were_VBD approved_VVN by_PRP the_AT0 University_NN1 of_PRF Colorado_NP0 Anschutz_NP0 Medical_AJ0 Campus_NN1 Institutional_AJ0 Animal_NN1 Care_NN1 and_CJC Use_NN1 Committee_NN1 ._SENT Sema7atm1Alk_NP0 /_PUN J_NP0 mice_NN2 (_PUL a_AT0 generous_AJ0 gift_NN1 from_PRP Alex_NP0 Kolodkin_NP0 at_PRP John_NP0 's_POS Hopkins_NP0 University_NN1 )_PUR and_CJC C57Bl_NP0 /_PUN 6_CRD (_PUL Jax_NP0 )_PUR were_VBD housed_VVN and_CJC bred_VVN as_CJS previously_AV0 described_VVN ._SENT Briefly_AV0 ,_PUN C57Bl_NP0 /_PUN 6_CRD SEMA7Atm1Alk_NN1 females_NN2 were_VBD bred_VVN with_PRP WT_NN1 C57Bl_NN1 /_PUN 6_CRD males_NN2 to_TO0 induce_VVI pregnancy_NN1 ,_PUN lactation_NN1 ,_PUN and_CJC postpartum_NN1 involution_NN1 ._SENT Pup_NN1 numbers_NN2 were_VBD normalized_VVN to_PRP 6_CRD –_PUN 8_CRD per_PRP dam_NN1 after_PRP birth_NN1 to_TO0 ensure_VVI for_PRP adequate_AJ0 lactation_NN1 ._SENT Postpartum_NN1 involution_NN1 was_VBD initiated_VVN by_PRP forced_AJ0 weaning_AJ0 of_PRF pups_NN2 at_PRP the_AT0 peak_NN1 of_PRF lactation_NN1 (_PUL L10-L12_CRD )_PUR ._SENT #4_CRD right_AJ0 and_CJC left_AJ0 mammary_AJ0 glands_NN2 were_VBD harvested_VVN from_PRP age-matched_AJ0 nulliparous_AJ0 animals_NN2 and_CJC postpartum_NN1 dams_NN2 at_PRP involution_NN1 days_NN2 1_CRD ,_PUN 2_CRD ,_PUN 4_CRD ,_PUN 6_CRD ,_PUN 8_CRD ,_PUN 10_CRD ,_PUN 14_CRD ,_PUN and_CJC 28._CRD For_PRP flow_NN1 cytometry_NN1 ,_PUN inguinal_AJ0 lymph_NN1 nodes_NN2 were_VBD removed_VVN prior_AJ0 to_PRP tissue_NN1 digestion_NN1 ._SENT #3_CRD mammary_AJ0 glands_NN2 were_VBD also_AV0 harvested_VVN ,_PUN formalin-fixed_AJ0 ,_PUN paraffin-embedded_NN1 ,_PUN and_CJC 5_CRD μm_AJ0 sections_NN2 generated_VVN for_PRP histologic_AJ0 /_PUN immunohistochemical_AJ0 analaysis_NN1 ._SENT See_VVB Supplemental_AJ0 Table_NN1 1_CRD for_PRP animal_NN1 study_NN1 sample_NN1 sizes_NN2 ._SENT 
In_PRP vitro_NN1 survival_NN1 assays_NN2 
MCF10A_NP0 and_CJC MCF12A_NP0 cells_NN2 were_VBD cultured_VVN with_PRP 200_CRD ng_NN0 /_PUN ml_NN0 purified_VVD SEMA7A_NP0 or_CJC PBS_NP0 vehicle_NN1 control_NN1 in_PRP adherent_NN1 (_PUL tissue_NN1 culture_NN1 plastic_NN1 )_PUR or_CJC forced_AJ0 suspension_NN1 conditions_NN2 (_PUL ultra-low_AJ0 attachment_NN1 plates_NN2 (_PUL Corning_NP0 ,_PUN Corning_NP0 ,_PUN NY_NP0 ,_PUN USA_NP0 :_PUN #3473_CRD )_PUR )_PUR ._SENT Cells_NN2 were_VBD seeded_VVN at_PRP a_AT0 density_NN1 of_PRF 1000_CRD cells_NN2 /_PUN well_AV0 in_PRP a_AT0 96-well_AJ0 plate_NN1 plus_PRP media_NN0 only_AJ0 wells_NN2 as_PRP controls_NN2 ._SENT Cell_NN1 death_NN1 was_VBD measured_VVN 24_CRD h_ZZ0 post_NN1 seeding_VVG via_PRP luminescence_NN1 using_VVG the_AT0 Caspase_NP0 Glo_NN1 assay_NN1 (_PUL Promega_NP0 ,_PUN Madison_NP0 ,_PUN WI_NP0 ,_PUN USA_NP0 :_PUN #G8090_CRD )_PUR ._SENT Annexin-V_NP0 /_PUN 7AAD_AJ0 staining_NN1 (_PUL Biolegend_NP0 ,_PUN San_NP0 Diego_NP0 ,_PUN CA_NP0 ,_PUN USA_NP0 :_PUN #640930_CRD )_PUR and_CJC pAKTS473_NP0 (_PUL ThermoFisher_NP0 :_PUN Waltham_NP0 ,_PUN MA_NP0 ,_PUN USA_NP0 ,_PUN #17-9715-42_CRD )_PUR were_VBD also_AV0 used_VVN to_TO0 confirm_VVI cell_NN1 viability_NN1 by_PRP flow_NN1 cytometry_NN1 ._SENT See_VVB flow_NN1 cytometry_NN1 methods_NN2 for_PRP staining_AJ0 protocol_NN1 ._SENT Function_NN1 blocking_VVG antibodies_NN2 for_PRP b1_NN1 (_PUL CD29-_NN1 9EG7_CRD ;_PUN BD_NP0 Biosciences_NP0 :_PUN #550531_CRD )_PUR and_CJC a6_AJ0 integrin_NN1 (_PUL ThermoFisher_NP0 :_PUN #14-0495-82_CRD )_PUR were_VBD used_VVN to_TO0 disrupt_VVI integrin_NN1 signaling_VVG in_PRP the_AT0 presence_NN1 of_PRF SEMA7A_NN1 or_CJC as_PRP antibody_NN1 alone_AV0 controls_VVZ to_TO0 determine_VVI off-target_AJ0 effects_NN2 ._SENT IgG_NN1 controls_NN2 were_VBD also_AV0 used_VVN ._SENT 9EG7_CRD was_VBD used_VVN at_PRP a_AT0 concentration_NN1 of_PRF 0.6_CRD μg_NN1 /_PUN ml_NN0 while_CJS GoH3_NP0 was_VBD used_VVN at_PRP a_AT0 concentration_NN1 of_PRF 100_CRD μg_NN1 /_PUN mL_NN0 for_PRP inhibition_NN1 studies_NN2 consistent_AJ0 with_PRP previous_AJ0 reports_NN2 ._SENT Cells_NN2 were_VBD cultured_VVN with_PRP anti-integrin_AJ0 inhibitors_NN2 at_PRP time_NN1 of_PRF seeding_NN1 ._SENT 
Label_NN1 retention_NN1 stem_NN1 cell_NN1 assay_NN1 
MCF10A_NP0 and_CJC MCF12A_NP0 cells_NN2 were_VBD labeled_VVN with_PRP CellTrace_NP0 Violet_NP0 (_PUL ThermoFisher_NP0 :_PUN #C34557_CRD )_PUR for_PRP 20_CRD min_NN0 at_PRP 37_CRD °_NN0 C_ZZ0 according_VVG to_PRP the_AT0 manufacturer_NN1 's_POS instructions_NN2 ._SENT A_AT0 sample_NN1 of_PRF labeled_AJ0 cells_NN2 was_VBD analyzed_VVN by_PRP flow_NN1 cytometry_NN1 to_TO0 determine_VVI labeling_NN1 efficiency_NN1 and_CJC fluorescent_AJ0 intensity_NN1 at_PRP time_NN1 of_PRF seeding_NN1 (_PUL Supplemental_AJ0 Fig._NN1 1_CRD )_PUR Single_AJ0 cells_NN2 were_VBD seeded_VVN at_PRP a_AT0 density_NN1 of_PRF 4000_CRD cells_NN2 /_PUN well_AV0 in_PRP ultra-low_AJ0 attachment_NN1 24-well_NN1 plates_NN2 to_TO0 induce_VVI mammosphere_NN1 formation_NN1 ._SENT Cells_NN2 were_VBD cultured_VVN in_PRP normal_AJ0 culture_NN1 media_NN0 for_PRP 7_CRD days_NN2 and_CJC were_VBD subsequently_AV0 counted_VVN ,_PUN dissociated_VVD enzymatically_AV0 in_PRP Accutase_NN1 ,_PUN (_PUL Stem_VVB Cell_NN1 Technologies_NN2 ,_PUN Vancouver_NP0 ,_PUN Canada_NP0 :_PUN #07922_CRD )_PUR ,_PUN and_CJC analyzed_VVN by_PRP flow_NN1 cytometry_NN1 ._SENT See_VVB flow_NN1 cytometry_NN1 methods_NN2 for_PRP detailed_AJ0 information_NN1 ._SENT 
Paclitaxel_VVB resistance_NN1 studies_NN2 
replaced-dns_NN2 cells_NN2 were_VBD treated_VVN with_PRP 100_CRD nM_NN1 paclitaxel_NN1 (_PUL Millipore_NP0 Sigma_NN1 ,_PUN Burling_NP0 ,_PUN MA_NP0 ,_PUN USA_NP0 #T7402-5MG_NP0 )_PUR for_PRP 24_CRD h_ZZ0 prior_AJ0 to_PRP analysis_NN1 by_PRP flow_NN1 cytometry_NN1 ._SENT Dose_NN1 was_VBD determined_VVN experimentally_AV0 based_VVN on_PRP cell_NN1 viability_NN1 and_CJC cancer_NN1 stem_NN1 cell_NN1 enrichment_NN1 ._SENT Drug_NN1 resistance_NN1 was_VBD measured_VVN by_PRP label-retention_NN1 and_CJC mammosphere_NN1 formation_NN1 as_CJS described_VVN above_AV0 and_CJC %_NN0 CD44+_NN1 (_PUL Biolegend_NP0 :_PUN #103018_CRD )_PUR CD24-_NN1 (_PUL Biolegend_NP0 :_PUN #311104_CRD )_PUR of_PRF singlets_NN2 was_VBD used_VVN as_PRP a_AT0 molecular_AJ0 definition_NN1 of_PRF drug-resistant_AJ0 stem_NN1 cells_NN2 ._SENT See_VVB label_NN1 retention_NN1 stem_NN1 cell_NN1 assay_NN1 and_CJC flow_NN1 cytometry_NN1 methods_NN2 for_PRP additional_AJ0 details_NN2 ._SENT 
Flow_NN1 cytometry_NN1 
Single_AJ0 cell_NN1 suspensions_NN2 were_VBD generated_VVN from_PRP cultured_AJ0 cells_NN2 via_PRP enzymatic_AJ0 harvest_NN1 with_PRP Accutase_NN1 ._SENT Mouse_NN1 mammary_AJ0 tissues_NN2 were_VBD minced_VVN with_PRP scalpels_NN2 ,_PUN digested_VVN in_PRP Click_NN1 's_POS media_NN0 containing_VVG 500_CRD units_NN2 /_PUN mL_NN0 Collagenase_NN1 II_CRD (_PUL Worthington_NP0 ,_PUN Columbus_NP0 ,_PUN OH_ITJ ,_PUN USA_NP0 ;_PUN LS004174_CRD )_PUR and_CJC Collagenase_NN1 IV_CRD (_PUL Worthington_NP0 ,_PUN LS004186_CRD )_PUR and_CJC 20_CRD μg_NN1 /_PUN mL_NN0 DNAse_NN1 (_PUL Worthington_NP0 ,_PUN LS002004_CRD )_PUR for_PRP 1_CRD h_ZZ0 at_PRP 37_CRD °_NN0 C_ZZ0 with_PRP occasional_AJ0 trituration_NN1 and_CJC strained_VVD through_PRP 70um_AJ0 filters_NN2 to_TO0 generate_VVI single_AJ0 cell_NN1 suspensions_NN2 ._SENT Single_AJ0 cells_NN2 were_VBD stained_VVN at_PRP 4_CRD °_NN0 C_ZZ0 for_PRP 30_CRD min_NN0 and_CJC washed_VVD with_PRP PBS_NP0 +_NN1 2_CRD %_NN0 FBS_NN2 ._SENT Cells_NN2 were_VBD filtered_VVN through_PRP 30_CRD μm_AJ0 filters_NN2 ,_PUN diluted_VVN to_PRP <_NP0 106_CRD cells_NN2 /_PUN ml_NN0 ,_PUN and_CJC analyzed_VVN with_PRP the_AT0 ZE5-YETI_NP0 flow_NN1 cytometer_NN1 ._SENT Fixation_NN1 /_PUN Permeabilization_NN1 kit_NN1 (_PUL BD_NP0 Biosciences_NP0 ,_PUN Franklin_NP0 Lakes_NP0 ,_PUN NJ_NP0 ,_PUN USA_NP0 :_PUN #554715_CRD )_PUR was_VBD used_VVN for_PRP intracellular_AJ0 antigen_NN1 staining_NN1 ._SENT Data_NN0 were_VBD analyzed_VVN with_PRP Kaluza_NP0 software_NN1 ._SENT Single_AJ0 stain_NN1 ,_PUN FMO_NP0 ,_PUN and_CJC unstained_AJ0 controls_NN2 were_VBD used_VVN to_TO0 set_VVI gates_NN2 ._SENT Single_AJ0 cells_NN2 were_VBD lineage_NN1 depleted_VVN using_VVG CD45-_NN1 (_PUL BD_NP0 Biosciences_NP0 :_PUN #564279_CRD )_PUR /_PUN CD31-_NP0 (_PUL BD_NP0 Biosciences_NP0 :_PUN #745436_CRD )_PUR gating_NN1 ._SENT Full_AJ0 gating_NN1 strategy_NN1 is_VBZ included_VVN as_PRP Supplemental_AJ0 Fig._NN1 2._CRD 
Immunohistochemistry_NN1 
Tissues_NN2 were_VBD formalin-fixed_AJ0 and_CJC paraffin-embedded_NN1 as_CJS previously_AV0 described_VVN ._SENT Hematoxylin_NN1 and_CJC Eosin_NN1 staining_NN1 was_VBD used_VVN to_TO0 define_VVI morphological_AJ0 features_NN2 ._SENT Antigen_NN1 retrieval_NN1 was_VBD performed_VVN using_VVG target_NN1 retrieval_NN1 solution_NN1 (_PUL Dako_NP0 ,_PUN Glostrup_NP0 ,_PUN Denmark_NP0 ;_PUN #S1699_CRD )_PUR for_PRP CC3_CRD (_PUL Cell_NN1 Signaling_AJ0 Technologies_NN2 ,_PUN Danvers_NP0 ,_PUN MA_NP0 ,_PUN USA_NP0 ;_PUN #9661_CRD )_PUR and_CJC Perilipin_NP0 (_PUL Cell_NN1 Signaling_AJ0 :_PUN #3470_CRD )_PUR or_CJC (_PUL EDTA_NP0 Dako_NP0 cat#_NN1 S2367_CRD )_PUR for_PRP pSTAT3_CRD (_PUL Cell_NN1 Signaling_AJ0 Technologies_NN2 ;_PUN #9145_CRD )_PUR ._SENT ImmPRESS_NN1 polymer_NN1 anti-rabbit_NN1 IgG_NN1 secondary_AJ0 reagent_NN1 (_PUL Vector_NN1 Labs_NN2 ,_PUN San_NP0 Francisco_NP0 ,_PUN CA_NP0 ,_PUN USA_NP0 t#MP-7401-15_CRD )_PUR was_VBD used_VVN for_PRP secondary_AJ0 staining_NN1 of_PRF CC3_CRD and_CJC pSTAT3_CRD stained_AJ0 tissues_NN2 and_CJC anti-rabbit_AJ0 secondary_NN1 (_PUL Dako_NP0 #K4003_CRD )_PUR for_PRP perilipin_AJ0 stains_NN2 ._SENT DAB_NP0 (_PUL Vector_NN1 Labs_NN2 ;_PUN #SK-4105_CRD )_PUR and_CJC counterstaining_NN1 were_VBD performed_VVN with_PRP hematoxylin_NP0 (_PUL Vector_NN1 Labs_NN2 #H-3401_CRD )_PUR ._SENT Alveolar_AJ0 area_NN1 was_VBD measured_VVN on_PRP H_NP0 &_NP0 E-stained_AJ0 tissues_NN2 in_PRP ImageJ_NP0 on_PRP 10_CRD alveoli_NN2 /_PUN field_NN1 ;_PUN 5_CRD fields_NN2 /_PUN tissue_NN1 ._SENT 10_CRD fields_NN2 /_PUN tissue_NN1 were_VBD analyzed_VVN for_PRP positive_AJ0 CC3_CRD ,_PUN pSTAT3_CRD ,_PUN and_CJC perilipin_AJ0 stained_AJ0 tissues_NN2 using_VVG CellSense_AJ0 Dimension_NN1 software_NN1 count_NN1 and_CJC measure_NN1 feature_NN1 on_PRP regions_NN2 of_PRF interest_NN1 ._SENT 
Data_NN0 mining_NN1 
Analysis_NN1 of_PRF SEMA7A_NN1 in_PRP breast_NN1 cancer_NN1 versus_PRP normal_AJ0 was_VBD performed_VVN on_PRP replaced-url_NN1 by_PRP selecting_VVG breast_NN1 cancer_NN1 samples_NN2 from_PRP The_AT0 Cancer_NN1 Genome_NN1 Atlas_NN1 (_PUL TCGA_NP0 )_PUR ._SENT Five-year_AJ0 relapse-free_AJ0 survival_NN1 (_PUL RFS_NP0 )_PUR curves_NN2 for_PRP Sema7a_NP0 (_PUL Affy_AJ0 ID_NN1 :_PUN 230345_at_NP0 )_PUR and_CJC Itga6_NP0 (_PUL Affy_AJ0 ID_NN1 :_PUN 201656_at_NN1 )_PUR were_VBD generated_VVN in_PRP KM_NN0 plot_NN1 using_VVG default_NN1 parameters_NN2 and_CJC no_AT0 restrictions_NN2 ._SENT Patients_NN2 split_VVN by_PRP median_AJ0 survival_NN1 ._SENT For_PRP multi-gene_AJ0 analyses_NN2 ,_PUN genes_NN2 were_VBD filtered_VVN by_PRP median_AJ0 expression_NN1 ._SENT 
Statistical_AJ0 analysis_NN1 
Unpaired_AJ0 t-test_NN1 and_CJC one-way_AJ0 ANOVA_NN1 were_VBD performed_VVN in_PRP GraphPad_NN1 Prism_NN1 ._SENT Nonparametric_AJ0 equivalent_AJ0 analyses_NN2 were_VBD used_VVN for_PRP samples_NN2 with_PRP uneven_AJ0 distribution_NN1 or_CJC unequal_AJ0 standard_AJ0 deviations_NN2 ._SENT p_ZZ0 values_NN2 of_PRF <_NP0 0.05_CRD were_VBD considered_VVN significant_AJ0 ._SENT Error_NN1 bars_NN2 represent_VVB mean_AJ0 ±_NN0 standard_AJ0 error_NN1 of_PRF the_AT0 mean_NN1 ._SENT All_DT0 in_PRP vivo_NN1 studies_NN2 were_VBD replicated_VVN in_PRP at_PRP least_AV0 three_CRD mice_NN2 and_CJC in_PRP vitro_NN1 studies_NN2 performed_VVN in_PRP technical_AJ0 and_CJC biological_AJ0 triplicate_NN1 with_PRP representative_AJ0 data_NN0 presented_VVN ._SENT 
Results_NN2 
Semaphorin-7a_NP0 promotes_VVZ anoikis_NN1 resistance_NN1 in_PRP normal_AJ0 MECs_NN2 
To_TO0 identify_VVI whether_CJS SEMA7A_NN1 promotes_VVZ survival_NN1 of_PRF normal_AJ0 MECs_NN2 we_PNP utilized_VVD two_CRD methods_NN2 :_PUN transfection_NN1 of_PRF FC-tagged_AJ0 SEMA7A_NN1 into_PRP immortalized_VVN human_AJ0 (_PUL MCF10A_NP0 )_PUR cells_NN2 (_PUL Supplemental_AJ0 Fig._NN1 3_CRD )_PUR and_CJC addition_NN1 of_PRF exogenous_AJ0 SEMA7A_NN1 ,_PUN which_DTQ we_PNP purified_VVD from_PRP FC-tagged_AJ0 SEMA7A_NN1 expressing_VVG cells_NN2 ,_PUN to_PRP wild-type_AJ0 MCF10A_NP0 and_CJC MCF12A_NP0 cells_NN2 —_PUN another_DT0 non-transformed_AJ0 MEC_NN1 line_NN1 ._SENT We_PNP found_VVD that_CJT genetic_AJ0 SEMA7A_NN1 overexpression_NN1 resulted_VVN in_PRP resistance_NN1 to_PRP cell_NN1 death_NN1 ,_PUN as_CJS measured_VVN by_PRP cleaved_AJ0 caspase_NN1 3_CRD /_PUN 7_CRD ,_PUN in_PRP a_AT0 forced_AJ0 suspension_NN1 assay_NN1 ,_PUN but_CJC not_XX0 in_PRP attached_AJ0 conditions_NN2 suggesting_VVG that_DT0 SEMA7A_NN1 specifically_AV0 mediates_VVZ resistance_NN1 to_PRP cell_NN1 death_NN1 by_PRP anoikis_NP0 (_PUL Fig._NN1 1a_CRD )_PUR ._SENT To_TO0 determine_VVI the_AT0 contribution_NN1 of_PRF endogenous_AJ0 SEMA7A_NN1 protein_NN1 to_PRP anoikis_NN1 resistance_NN1 ,_PUN we_PNP measured_VVD SEMA7A_NP0 +_NP0 MECs_NN2 by_PRP flow_NN1 over_PRP time_NN1 in_PRP attached_AJ0 and_CJC suspended_AJ0 conditions_NN2 and_CJC found_VVD that_CJT SEMA7A_NP0 +_NP0 MECs_NN2 in_PRP attached_AJ0 conditions_NN2 remain_VVB unchanged_AJ0 while_CJS SEMA7A_NP0 +_NP0 cells_NN2 become_VVB significantly_AV0 enriched_AJ0 as_CJS early_AV0 as_CJS 24_CRD h_ZZ0 in_PRP detached_AJ0 conditions_NN2 (_PUL Fig._NN1 1b_CRD )_PUR ._SENT We_PNP also_AV0 show_VVB that_DT0 stimulation_NN1 with_PRP exogenous_AJ0 purified_AJ0 SEMA7A_NN1 protein_NN1 in_PRP both_AV0 MCF10A_NP0 and_CJC MCF12A_NP0 cells_NN2 is_VBZ sufficient_AJ0 to_TO0 significantly_AV0 decrease_VVI cell_NN1 death_NN1 and_CJC inhibition_NN1 of_PRF a_AT0 known_AJ0 SEMA7A_NN1 receptor_NN1 ,_PUN β1-integrin_NN1 ,_PUN significantly_AV0 abrogated_VVN SEMA7A-mediated_NN1 anoikis_NN1 resistance_NN1 (_PUL Fig._NN1 1c_NN1 and_CJC Supplemental_AJ0 Figs._NN2 4_CRD and_CJC 5_CRD )_PUR ._SENT 
